SMART MODE:

NORMAL GENOMIC

• Xu X, Nakazawa N, Yanagida M
• Condensin HEAT subunits required for DNA repair, kinetochore/centromere function and ploidy maintenance in fission yeast.
• PLoS One. 2015; 10: 119347-119347
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• Condensin, a central player in eukaryotic chromosomal dynamics, contains five evolutionarily-conserved subunits. Two SMC (structural maintenance of chromosomes) subunits contain ATPase, hinge, and coiled-coil domains. One non-SMC subunit is similar to bacterial kleisin, and two other non-SMC subunits contain HEAT (similar to armadillo) repeats. Here we report isolation and characterization of 21 fission yeast (Schizosaccharomyces pombe) mutants for three non-SMC subunits, created using error-prone mutagenesis that resulted in single-amino acid substitutions. Beside condensation, segregation, and DNA repair defects, similar to those observed in previously isolated SMC and cnd2 mutants, novel phenotypes were observed for mutants of HEAT-repeats containing Cnd1 and Cnd3 subunits. cnd3-L269P is hypersensitive to the microtubule poison, thiabendazole, revealing defects in kinetochore/centromere and spindle assembly checkpoints. Three cnd1 and three cnd3 mutants increased cell size and doubled DNA content, thereby eliminating the haploid state. Five of these mutations reside in helix B of HEAT repeats. Two non-SMC condensin subunits, Cnd1 and Cnd3, are thus implicated in ploidy maintenance.

• Kang H et al.
• Sex comb on midleg (Scm) is a functional link between PcG-repressive complexes in Drosophila.
• Genes Dev. 2015; 29: 1136-50
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• The Polycomb group (PcG) proteins are key regulators of development in Drosophila and are strongly implicated in human health and disease. How PcG complexes form repressive chromatin domains remains unclear. Using cross-linked affinity purifications of BioTAP-Polycomb (Pc) or BioTAP-Enhancer of zeste [E(z)], we captured all PcG-repressive complex 1 (PRC1) or PRC2 core components and Sex comb on midleg (Scm) as the only protein strongly enriched with both complexes. Although previously not linked to PRC2, we confirmed direct binding of Scm and PRC2 using recombinant protein expression and colocalization of Scm with PRC1, PRC2, and H3K27me3 in embryos and cultured cells using ChIP-seq (chromatin immunoprecipitation [ChIP] combined with deep sequencing). Furthermore, we found that RNAi knockdown of Scm and overexpression of the dominant-negative Scm-SAM (sterile alpha motif) domain both affected the binding pattern of E(z) on polytene chromosomes. Aberrant localization of the Scm-SAM domain in long contiguous regions on polytene chromosomes revealed its independent ability to spread on chromatin, consistent with its previously described ability to oligomerize in vitro. Pull-downs of BioTAP-Scm captured PRC1 and PRC2 and additional repressive complexes, including PhoRC, LINT, and CtBP. We propose that Scm is a key mediator connecting PRC1, PRC2, and transcriptional silencing. Combined with previous structural and genetic analyses, our results strongly suggest that Scm coordinates PcG complexes and polymerizes to produce broad domains of PcG silencing.

• Barysz H et al.
• Three-dimensional topology of the SMC2/SMC4 subcomplex from chicken condensin I revealed by cross-linking and molecular modelling.
• Open Biol. 2015; 5: 150005-150005
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• SMC proteins are essential components of three protein complexes that are important for chromosome structure and function. The cohesin complex holds replicated sister chromatids together, whereas the condensin complex has an essential role in mitotic chromosome architecture. Both are involved in interphase genome organization. SMC-containing complexes are large (more than 650 kDa for condensin) and contain long anti-parallel coiled-coils. They are thus difficult subjects for conventional crystallographic and electron cryomicroscopic studies. Here, we have used amino acid-selective cross-linking and mass spectrometry combined with structure prediction to develop a full-length molecular draft three-dimensional structure of the SMC2/SMC4 dimeric backbone of chicken condensin. We assembled homology-based molecular models of the globular heads and hinges with the lengthy coiled-coils modelled in fragments, using numerous high-confidence cross-links and accounting for potential irregularities. Our experiments reveal that isolated condensin complexes can exist with their coiled-coil segments closely apposed to one another along their lengths and define the relative spatial alignment of the two anti-parallel coils. The centres of the coiled-coils can also approach one another closely in situ in mitotic chromosomes. In addition to revealing structural information, our cross-linking data suggest that both H2A and H4 may have roles in condensin interactions with chromatin.

• Gerace E, Moazed D
• Coimmunoprecipitation of proteins from yeast.
• Methods Enzymol. 2014; 541: 13-26
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• This protocol outlines a procedure for testing whether two proteins interact. A target protein will be immunoprecipitated using an antibody that recognizes it (or a tagged version of the protein). The immunoprecipitated material will be separated by SDS-PAGE and analyzed by Western blotting to assess the presence of a candidate interacting protein(s).

• Robellet X et al.
• A genetic screen for functional partners of condensin in fission yeast.
• G3 (Bethesda). 2014; 4: 373-81
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• Mitotic chromosome condensation is a prerequisite for the accurate segregation of chromosomes during cell division, and the conserved condensin complex a central player of this process. However, how condensin binds chromatin and shapes mitotic chromosomes remain poorly understood. Recent genome-wide binding studies showing that in most species condensin is enriched near highly expressed genes suggest a conserved link between condensin occupancy and high transcription rates. To gain insight into the mechanisms of condensin binding and mitotic chromosome condensation, we searched for factors that collaborate with condensin through a synthetic lethal genetic screen in the fission yeast Schizosaccharomyces pombe. We isolated novel mutations affecting condensin, as well as mutations in four genes not previously implicated in mitotic chromosome condensation in fission yeast. These mutations cause chromosome segregation defects similar to those provoked by defects in condensation. We also identified a suppressor of the cut3-477 condensin mutation, which largely rescued chromosome segregation during anaphase. Remarkably, of the five genes identified in this study, four encode transcription co-factors. Our results therefore provide strong additional evidence for a functional connection between chromosome condensation and transcription.

• Slingsby C, Clark AR
• Flexible nanoassembly for sequestering non-native proteins.
• Structure. 2013; 21: 193-4
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• A crystal structure of a yeast small heat shock protein reported by Hanazono and colleagues in this issue of Structure reveals the versatility of the alpha-crystallin domain dimer for building assemblies of different size and symmetry. The domains assemble into a vessel filled with hydrophobic sequence extensions enriched with phenylalanines.

• Copsey A et al.
• Smc5/6 coordinates formation and resolution of joint molecules with chromosome morphology to ensure meiotic divisions.
• PLoS Genet. 2013; 9: 1004071-1004071
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• During meiosis, Structural Maintenance of Chromosome (SMC) complexes underpin two fundamental features of meiosis: homologous recombination and chromosome segregation. While meiotic functions of the cohesin and condensin complexes have been delineated, the role of the third SMC complex, Smc5/6, remains enigmatic. Here we identify specific, essential meiotic functions for the Smc5/6 complex in homologous recombination and the regulation of cohesin. We show that Smc5/6 is enriched at centromeres and cohesin-association sites where it regulates sister-chromatid cohesion and the timely removal of cohesin from chromosomal arms, respectively. Smc5/6 also localizes to recombination hotspots, where it promotes normal formation and resolution of a subset of joint-molecule intermediates. In this regard, Smc5/6 functions independently of the major crossover pathway defined by the MutLgamma complex. Furthermore, we show that Smc5/6 is required for stable chromosomal localization of the XPF-family endonuclease, Mus81-Mms4(Eme1). Our data suggest that the Smc5/6 complex is required for specific recombination and chromosomal processes throughout meiosis and that in its absence, attempts at cell division with unresolved joint molecules and residual cohesin lead to severe recombination-induced meiotic catastrophe.

• Xaver M, Huang L, Chen D, Klein F
• Smc5/6-Mms21 prevents and eliminates inappropriate recombination intermediates in meiosis.
• PLoS Genet. 2013; 9: 1004067-1004067
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• Repairing broken chromosomes via joint molecule (JM) intermediates is hazardous and therefore strictly controlled in most organisms. Also in budding yeast meiosis, where production of enough crossovers via JMs is imperative, only a subset of DNA breaks are repaired via JMs, closely regulated by the ZMM pathway. The other breaks are repaired to non-crossovers, avoiding JM formation, through pathways that require the BLM/Sgs1 helicase. "Rogue" JMs that escape the ZMM pathway and BLM/Sgs1 are eliminated before metaphase by resolvases like Mus81-Mms4 to prevent chromosome nondisjunction. Here, we report the requirement of Smc5/6-Mms21 for antagonizing rogue JMs via two mechanisms; destabilizing early intermediates and resolving JMs. Elimination of the Mms21 SUMO E3-ligase domain leads to transient JM accumulation, depending on Mus81-Mms4 for resolution. Absence of Smc6 leads to persistent rogue JMs accumulation, preventing chromatin separation. We propose that the Smc5/6-Mms21 complex antagonizes toxic JMs by coordinating helicases and resolvases at D-Loops and HJs, respectively.

• Xu M, Chang YP, Chen XS
• Expression, purification and biochemical characterization of Schizosaccharomyces pombe Mcm4, 6 and 7.
• BMC Biochem. 2013; 14: 5-5
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• BACKGROUND: The hetero-hexamer of the eukaryotic minichromosome maintenance (MCM) proteins plays an essential role in replication of genomic DNA. The ring-shaped Mcm2-7 hexamers comprising one of each subunit show helicase activity in vitro, and form double-hexamers on DNA. The Mcm4/6/7 also forms a hexameric complex with helicase activity in vitro. RESULTS: We used an Escherichiai coli expression system to express various domains of Schizosaccharomyces pombe Mcm4, 6 and 7 in order to characterize their domain structure, oligomeric states, and possible inter-/intra-subunit interactions. We also successfully employed a co-expression system to express Mcm4/6/7 at the same time in Escherichiai coli, and have purified functional Mcm4/6/7 complex in a hexameric state in high yield and purity, providing a means for generating large quantity of proteins for future structural and biochemical studies. CONCLUSIONS: Based on our results and those of others, models were proposed for the subunit arrangement and architecture of both the Mcm4/6/7 hexamer and the Mcm2-7 double-hexamer.

• Bustard DE et al.
• During replication stress, non-SMC element 5 (NSE5) is required for Smc5/6 protein complex functionality at stalled forks.
• J Biol Chem. 2012; 287: 11374-83
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• The Smc5/6 complex belongs to the SMC (structural maintenance of chromosomes) family, which also includes cohesin and condensin. In Saccharomyces cerevisiae, the Smc5/6 complex contains six essential non-Smc elements, Nse1-6. Very little is known about how these additional elements contribute to complex function except for Nse2/Mms21, which is an E3 small ubiquitin-like modifier (SUMO) ligase important for Smc5 sumoylation. Characterization of two temperature-sensitive mutants, nse5-ts1 and nse5-ts2, demonstrated the importance of Nse5 within the Smc5/6 complex for its stability and functionality at forks during hydroxyurea-induced replication stress. Both NSE5 alleles showed a marked reduction in Smc5 sumoylation to levels lower than those observed with mms21-11, a mutant of Mms21 that is deficient in SUMO ligase activity. However, a phenotypic comparison of nse5-ts1 and nse5-ts2 revealed a separation of importance between Smc5 sumoylation and the function of the Smc5/6 complex during replication. Only cells carrying the nse5-ts1 allele exhibited defects such as dissociation of the replisome from stalled forks, formation of fork-associated homologous recombination intermediates, and hydroxyurea sensitivity that is additive with mms21-11. These defects are attributed to a failure in Smc5/6 localization to forks in nse5-ts1 cells. Overall, these data support the premise that Nse5 is important for vital interactions between components within the Smc5/6 complex, and for its functionality during replication stress.

• Guerineau M, Kriz Z, Kozakova L, Bednarova K, Janos P, Palecek J
• Analysis of the Nse3/MAGE-binding domain of the Nse4/EID family proteins.
• PLoS One. 2012; 7: 35813-35813
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• BACKGROUND: The Nse1, Nse3 and Nse4 proteins form a tight sub-complex of the large SMC5-6 protein complex. hNSE3/MAGEG1, the mammalian ortholog of Nse3, is the founding member of the MAGE (melanoma-associated antigen) protein family and the Nse4 kleisin subunit is related to the EID (E1A-like inhibitor of differentiation) family of proteins. We have recently shown that human MAGE proteins can interact with NSE4/EID proteins through their characteristic conserved hydrophobic pocket. METHODOLOGY/PRINCIPAL FINDINGS: Using mutagenesis and protein-protein interaction analyses, we have identified a new Nse3/MAGE-binding domain (NMBD) of the Nse4/EID proteins. This short domain is located next to the Nse4 N-terminal kleisin motif and is conserved in all NSE4/EID proteins. The central amino acid residues of the human NSE4b/EID3 domain were essential for its binding to hNSE3/MAGEG1 in yeast two-hybrid assays suggesting they form the core of the binding domain. PEPSCAN ELISA measurements of the MAGEC2 binding affinity to EID2 mutant peptides showed that similar core residues contribute to the EID2-MAGEC2 interaction. In addition, the N-terminal extension of the EID2 binding domain took part in the EID2-MAGEC2 interaction. Finally, docking and molecular dynamic simulations enabled us to generate a structure model for EID2-MAGEC2. Combination of our experimental data and the structure modeling showed how the core helical region of the NSE4/EID domain binds into the conserved pocket characteristic of the MAGE protein family. CONCLUSIONS/SIGNIFICANCE: We have identified a new Nse4/EID conserved domain and characterized its binding to Nse3/MAGE proteins. The conservation and binding of the interacting surfaces suggest tight co-evolution of both Nse4/EID and Nse3/MAGE protein families.

• Stephan AK, Kliszczak M, Morrison CG
• The Nse2/Mms21 SUMO ligase of the Smc5/6 complex in the maintenance of genome stability.
• FEBS Lett. 2011; 585: 2907-13
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• There exist three highly-conserved structural maintenance of chromosomes (Smc) complexes that ensure genome stability during eukaryotic cell division. There are the well-characterized cohesin and condensin complexes and the third Smc complex, Smc5/6. Nse2/Mms21, a SUMO ligase, is a component of the Smc5/6 complex and recent data have indicated that Nse1 may function as a ubiquitin ligase. Smc5/6 regulates sister chromatid cohesion, homologous recombination and chromatin structure and conformation. This review examines the functions of Smc5/6 in DNA repair and the maintenance of genomic integrity and explores the roles of the associated SUMO and ubiquitin ligases. Recent findings have indicated that Smc5/6 may play a topological role in chromosome dynamics, which may help understand the complexity of its activities.

• Duan X, Holmes WB, Ye H
• Interaction mapping between Saccharomyces cerevisiae Smc5 and SUMO E3 ligase Mms21.
• Biochemistry. 2011; 50: 10182-8
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• The multisubunit Smc5-Smc6 holocomplex (Smc5/6) plays a critical role in chromosome stability maintenance, DNA replication, homologous recombination, and double-stranded DNA damage repair. Smc5 and Smc6 form the core of the holocomplex, along with six non-SMC elements, for which most functions are not yet understood. Mms21 (Nse2), the relatively well-studied subunit in Smc5/6, contains a SP-like-RING finger motif on the C-terminus and was identified as a SUMO E3 ligase. Deletion of Mms21 is lethal; however, while deficient in DNA damage repair, SUMO ligase mutants remain viable. These functions of Mms21 in Smc5/6 are hard to address without understanding the interaction between Smc5 and Mms21. Previously, we systematically examined the architecture of Saccharomyces cerevisiae Smc5/6 and, using yeast two-hybrid methods, found that Mms21 interacts with the coiled-coil of Smc5. Later, crystallographic studies revealed the molecular arrangement of Mms21 with Smc5/6. For this study, we use a combination of limited proteolysis, mass spectrometry, and N-terminal sequencing to precisely define the interaction region of Smc5 with Mms21. In addition, using isothermal titration calorimetry, we find that Mms21 interacts with Smc5 in a 1:1 ratio with a K(d) of 0.68 muM. This combination of methods would be useful in examining the structure of any large multiprotein complex.

• Yuan J, Chen J
• The role of the human SWI5-MEI5 complex in homologous recombination repair.
• J Biol Chem. 2011; 286: 9888-93
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• The Swi5-Mei5 complex and its homologues are involved in specialized recombination pathways in budding and fission yeasts. Although the fission yeast homologue Swi5-Sfr1 is critical for homologous recombination repair, the budding yeast counterpart Sae3-Mei5 is meiosis-specific, interacts with Dmc1, and promotes assembly of Dmc1 on meiotic chromosomes. Here, we identify and characterize the human SWI5-MEI5 (C9orf119-C10orf78) complex. We showed that SWI5 and MEI5 form a stable complex in vitro and in vivo. The C-terminal Swi5 domain of SWI5 and the middle coiled-coil region of MEI5 dictate this conserved interaction. In addition, SWI5-MEI5 directly interacts with RAD51 in vitro. Depletion of SWI5 or MEI5 in human cells causes defects in homologous recombination repair. Finally, SWI5- or MEI5-depleted cells display enhanced sensitivity to ionizing radiation, consistent with the role of this complex in HR repair. Our results suggest that human SWI5-MEI5 has an evolutionarily conserved function in homologous recombination repair.

• Nakazawa N, Mehrotra R, Ebe M, Yanagida M
• Condensin phosphorylated by the Aurora-B-like kinase Ark1 is continuously required until telophase in a mode distinct from Top2.
• J Cell Sci. 2011; 124: 1795-807
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• Condensin is a conserved protein complex that functions in chromosome condensation and segregation. It has not been previously unequivocally determined whether condensin is required throughout mitosis. Here, we examined whether Schizosaccharomyces pombe condensin continuously acts on chromosomes during mitosis and compared its role with that of DNA topoisomerase II (Top2). Using double mutants containing a temperature-sensitive allele of the condensin SMC2 subunit cut14 (cut14-208) or of top2, together with the cold-sensitive nda3-KM311 mutation (in beta-tubulin), temperature-shift experiments were performed. These experiments allowed inactivation of condensin or Top2 at various stages throughout mitosis, even after late anaphase. The results established that mitotic chromosomes require condensin and Top2 throughout mitosis, even in telophase. We then showed that the Cnd2 subunit of condensin (also known as Barren) is the target subunit of Aurora-B-like kinase Ark1 and that Ark1-mediated phosphorylation of Cnd2 occurred throughout mitosis. The phosphorylation sites in Cnd2 were determined by mass spectrometry, and alanine and glutamate residue replacement mutant constructs for these sites were constructed. Alanine substitution mutants of Cnd2, which mimic the unphosphorylated protein, exhibited broad mitotic defects, including at telophase, and overexpression of these constructs caused a severe dominant-negative effect. By contrast, glutamate substitution mutants, which mimic the phosphorylated protein, alleviated the segregation defect in Ark1-inhibited cells. In telophase, the condensin subunits in cut14-208 mutant accumulated in lumps that contained telomeric DNA and proteins that failed to segregate. Condensin might thus serve to keep the segregated chromosomes apart during telophase.

• Baudrimont A et al.
• A new thermosensitive smc-3 allele reveals involvement of cohesin in homologous recombination in C. elegans.
• PLoS One. 2011; 6: 24799-24799
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• The cohesin complex is required for the cohesion of sister chromatids and for correct segregation during mitosis and meiosis. Crossover recombination, together with cohesion, is essential for the disjunction of homologous chromosomes during the first meiotic division. Cohesin has been implicated in facilitating recombinational repair of DNA lesions via the sister chromatid. Here, we made use of a new temperature-sensitive mutation in the Caenorhabditis elegans SMC-3 protein to study the role of cohesin in the repair of DNA double-strand breaks (DSBs) and hence in meiotic crossing over. We report that attenuation of cohesin was associated with extensive SPO-11-dependent chromosome fragmentation, which is representative of unrepaired DSBs. We also found that attenuated cohesin likely increased the number of DSBs and eliminated the need of MRE-11 and RAD-50 for DSB formation in C. elegans, which suggests a role for the MRN complex in making cohesin-loaded chromatin susceptible to meiotic DSBs. Notably, in spite of largely intact sister chromatid cohesion, backup DSB repair via the sister chromatid was mostly impaired. We also found that weakened cohesins affected mitotic repair of DSBs by homologous recombination, whereas NHEJ repair was not affected. Our data suggest that recombinational DNA repair makes higher demands on cohesins than does chromosome segregation.

• Rai R et al.
• Small ubiquitin-related modifier ligase activity of Mms21 is required for maintenance of chromosome integrity during the unperturbed mitotic cell division cycle in Saccharomyces cerevisiae.
• J Biol Chem. 2011; 286: 14516-30
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• The SUMO ligase activity of Mms21/Nse2, a conserved member of the Smc5/6 complex, is required for resisting extrinsically induced genotoxic stress. We report that the Mms21 SUMO ligase activity is also required during the unchallenged mitotic cell cycle in Saccharomyces cerevisiae. SUMO ligase-defective cells were slow growing and spontaneously incurred DNA damage. These cells required caffeine-sensitive Mec1 kinase-dependent checkpoint signaling for survival even in the absence of extrinsically induced genotoxic stress. SUMO ligase-defective cells were sensitive to replication stress and displayed synthetic growth defects with DNA damage checkpoint-defective mutants such as mec1, rad9, and rad24. MMS21 SUMO ligase and mediator of replication checkpoint 1 gene (MRC1) were epistatic with respect to hydroxyurea-induced replication stress or methyl methanesulfonate-induced DNA damage sensitivity. Subjecting Mms21 SUMO ligase-deficient cells to transient replication stress resulted in enhancement of cell cycle progression defects such as mitotic delay and accumulation of hyperploid cells. Consistent with the spontaneous activation of the DNA damage checkpoint pathway observed in the Mms21-mediated sumoylation-deficient cells, enhanced frequency of chromosome breakage and loss was detected in these mutant cells. A mutation in the conserved cysteine 221 that is engaged in coordination of the zinc ion in Loop 2 of the Mms21 SPL-RING E3 ligase catalytic domain resulted in strong replication stress sensitivity and also conferred slow growth and Mec1 dependence to unchallenged mitotically dividing cells. Our findings establish Mms21-mediated sumoylation as a determinant of cell cycle progression and maintenance of chromosome integrity during the unperturbed mitotic cell division cycle in budding yeast.

• Farmer S, San-Segundo PA, Aragon L
• The Smc5-Smc6 complex is required to remove chromosome junctions in meiosis.
• PLoS One. 2011; 6: 20948-20948
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• Meiosis, a specialized cell division with a single cycle of DNA replication round and two consecutive rounds of nuclear segregation, allows for the exchange of genetic material between parental chromosomes and the formation of haploid gametes. The structural maintenance of chromosome (SMC) proteins aid manipulation of chromosome structures inside cells. Eukaryotic SMC complexes include cohesin, condensin and the Smc5-Smc6 complex. Meiotic roles have been discovered for cohesin and condensin. However, although Smc5-Smc6 is known to be required for successful meiotic divisions, the meiotic functions of the complex are not well understood. Here we show that the Smc5-Smc6 complex localizes to specific chromosome regions during meiotic prophase I. We report that meiotic cells lacking Smc5-Smc6 undergo catastrophic meiotic divisions as a consequence of unresolved linkages between chromosomes. Surprisingly, meiotic segregation defects are not rescued by abrogation of Spo11-induced meiotic recombination, indicating that at least some chromosome linkages in smc5-smc6 mutants originate from other cellular processes. These results demonstrate that, as in mitosis, Smc5-Smc6 is required to ensure proper chromosome segregation during meiosis by preventing aberrant recombination intermediates between homologous chromosomes.

• Hudson JJ et al.
• Interactions between the Nse3 and Nse4 components of the SMC5-6 complex identify evolutionarily conserved interactions between MAGE and EID Families.
• PLoS One. 2011; 6: 17270-17270
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• BACKGROUND: The SMC5-6 protein complex is involved in the cellular response to DNA damage. It is composed of 6-8 polypeptides, of which Nse1, Nse3 and Nse4 form a tight sub-complex. MAGEG1, the mammalian ortholog of Nse3, is the founding member of the MAGE (melanoma-associated antigen) protein family and Nse4 is related to the EID (E1A-like inhibitor of differentiation) family of transcriptional repressors. METHODOLOGY/PRINCIPAL FINDINGS: Using site-directed mutagenesis, protein-protein interaction analyses and molecular modelling, we have identified a conserved hydrophobic surface on the C-terminal domain of Nse3 that interacts with Nse4 and identified residues in its N-terminal domain that are essential for interaction with Nse1. We show that these interactions are conserved in the human orthologs. Furthermore, interaction of MAGEG1, the mammalian ortholog of Nse3, with NSE4b, one of the mammalian orthologs of Nse4, results in transcriptional co-activation of the nuclear receptor, steroidogenic factor 1 (SF1). In an examination of the evolutionary conservation of the Nse3-Nse4 interactions, we find that several MAGE proteins can interact with at least one of the NSE4/EID proteins. CONCLUSIONS/SIGNIFICANCE: We have found that, despite the evolutionary diversification of the MAGE family, the characteristic hydrophobic surface shared by all MAGE proteins from yeast to humans mediates its binding to NSE4/EID proteins. Our work provides new insights into the interactions, evolution and functions of the enigmatic MAGE proteins.

• Chavez A, George V, Agrawal V, Johnson FB
• Sumoylation and the structural maintenance of chromosomes (Smc) 5/6 complex slow senescence through recombination intermediate resolution.
• J Biol Chem. 2010; 285: 11922-30
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• Telomeres are repetitive nucleoprotein structures that cap the ends of chromosomes. Without telomerase, telomeres shorten with replication and eventually signal cell cycle arrest (cell senescence). Homologous recombination (HR)-based mechanisms slow senescence, and distinct HR mechanisms support the growth of the rare survivors of senescence. Here, we report novel roles for the post-translational modification of small ubiquitin-like modifier (SUMO) in regulating the rate of senescence in Saccharomyces cerevisiae telomerase mutants. We identify Mms21 as the relevant SUMO E3 ligase and demonstrate that cells lacking Mms21-dependent sumoylation accumulate HR intermediates selectively at telomeres during senescence. One target of Mms21-dependent sumoylation is the cohesin- and condensin-related Smc5-Smc6 complex (Smc5/6). We show that hypomorphic smc5 or smc6 alleles exhibit phenotypes similar to mms21 sumoylation-deficient mutants with regard to senescence and the accumulation of unresolved HR intermediates. Further, we provide evidence that Mms21 and Smc5/6 prevent aberrant recombination between sister telomeres and also globally facilitate resolution of sister chromatid HR intermediates.

• Bardhan A
• Many functions of the meiotic cohesin.
• Chromosome Res. 2010; 18: 909-24
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• Sister chromatids are held together from the time of their formation in S phase until they segregate in anaphase by the cohesin complex. In meiosis of most organisms, the mitotic Mcd1/Scc1/Rad21 subunit of the cohesin complex is largely replaced by its paralog named Rec8. This article reviews the specialized functions of Rec8 that are crucial for diverse aspects of chromosome dynamics in meiosis, and presents some speculations relating to meiotic chromosome organization.

• Kagawa W, Kurumizaka H
• From meiosis to postmeiotic events: uncovering the molecular roles of the meiosis-specific recombinase Dmc1.
• FEBS J. 2010; 277: 590-8
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• In meiosis, the accurate segregation of maternal and paternal chromosomes is accomplished by homologous recombination. A central player in meiotic recombination is the Dmc1 recombinase, a member of the RecA/Rad51 recombinase superfamily, which is widely conserved from viruses to humans. Dmc1 is a meiosis-specific protein that functions with the ubiquitously expressed homolog, the Rad51 recombinase, which is essential for both mitotic and meiotic recombination. Since its discovery, it has been speculated that Dmc1 is important for unique aspects of meiotic recombination. Understanding the distinctive properties of Dmc1, namely, the features that distinguish it from Rad51, will further clarify the mechanisms of meiotic recombination. Recent structural, biochemical, and genetic findings are now revealing the molecular mechanisms of Dmc1-mediated homologous recombination and its regulation by various recombination mediators.

• Kinoshita E, van der Linden E, Sanchez H, Wyman C
• RAD50, an SMC family member with multiple roles in DNA break repair: how does ATP affect function?
• Chromosome Res. 2009; 17: 277-88
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• The protein complex including Mre11, Rad50, and Nbs1 (MRN) functions in DNA double-strand break repair to recognize and process DNA ends as well as signal for cell cycle arrest. Amino acid sequence similarity and overall architecture make Rad50 a member of the structural maintenance of chromosome (SMC) protein family. Like SMC proteins, Rad50 function depends on ATP binding and hydrolysis. All current evidence indicates that ATP binding and hydrolysis cause architectural rearrangements in SMC protein complexes that are important for their functions in organizing DNA. In the case of the MRN complex, the functional significance of ATP binding and hydrolysis are not yet defined. Here we review the data on the ATP-dependent activities of MRN and their possible mechanistic significance. We present some speculation on the role of ATP for function of the MRN complex based on the similarities and differences in the molecular architecture of the Rad50-containing complexes and the SMC complexes condensin and cohesin.

• Lloyd J et al.
• A supramodular FHA/BRCT-repeat architecture mediates Nbs1 adaptor function in response to DNA damage.
• Cell. 2009; 139: 100-11
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• The Mre11/Rad50/Nbs1 protein complex plays central enzymatic and signaling roles in the DNA-damage response. Nuclease (Mre11) and scaffolding (Rad50) components of MRN have been extensively characterized, but the molecular basis of Nbs1 function has remained elusive. Here, we present a 2.3A crystal structure of the N-terminal region of fission yeast Nbs1, revealing an unusual but conserved architecture in which the FHA- and BRCT-repeat domains structurally coalesce. We demonstrate that diphosphorylated pSer-Asp-pThr-Asp motifs, recently identified as multicopy docking sites within Mdc1, are evolutionarily conserved Nbs1 binding targets. Furthermore, we show that similar phosphomotifs within Ctp1, the fission yeast ortholog of human CtIP, promote interactions with the Nbs1 FHA domain that are necessary for Ctp1-dependent resistance to DNA damage. Finally, we establish that human Nbs1 interactions with Mdc1 occur through both its FHA- and BRCT-repeat domains, suggesting how their structural and functional interdependence underpins Nbs1 adaptor functions in the DNA-damage response.

• Duan X, Ye H
• Purification, crystallization and preliminary X-ray crystallographic studies of the complex between Smc5 and the SUMO E3 ligase Mms21.
• Acta Crystallogr Sect F Struct Biol Cryst Commun. 2009; 65: 849-52
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• Smc5/6, a protein complex that belongs to the structural maintenance of chromosome (SMC) family, plays a key role in DNA replication, sister chromatid recombination and DNA damage repair. The complex contains eight subunits, including a SUMO E3 ligase Mms21 (Nse2). The activity of Mms21 is important for regulation of Smc5/6 in the response to DNA damage. Mms21 and the Mms21-binding region of Smc5 were overexpressed and purified individually in Escherichia coli with a C-terminal LEHHHHHH tag. The Mms21-Smc5 protein complex was crystallized. The diffraction of the crystals was improved greatly by glutaraldehyde treatment. X-ray diffraction data sets were collected to resolutions of 2.3 and 3.9 A from native and selenomethionine-derivative protein crystals, respectively. The crystals belonged to space group C222(1), with unit-cell parameters a = 47.465, b = 97.574, c = 249.215 A for the native crystals.

• Hudson DF, Marshall KM, Earnshaw WC
• Condensin: Architect of mitotic chromosomes.
• Chromosome Res. 2009; 17: 131-44
• Display abstract

• Condensin is a highly conserved pentameric complex consisting of two structural maintenance of chromosome (SMC) ATPase subunits and three auxiliary components. While initially regarded as a key driver of mitotic chromosome condensation, condensin is increasingly viewed as having a more subtle influence on chromosome architecture. The two condensin complexes are required to direct the correct folding and organization of chromosomes prior to anaphase and for keeping the chromosomes compact as they separate to the poles. This ancient complex is essential in mitosis and meiosis and has additional roles in gene regulation and DNA repair. The wide variety of biochemical and genetic tools available are gradually unravelling the numerous roles condensin plays during the cell cycle and shedding light on its mechanism of action.

• Miyabe I, Morishita T, Shinagawa H, Carr AM
• Schizosaccharomyces pombe Cds1Chk2 regulates homologous recombination at stalled replication forks through the phosphorylation of recombination protein Rad60.
• J Cell Sci. 2009; 122: 3638-43
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• The Schizosaccharomyces pombe rad60 gene is essential for cell growth and is involved in repairing DNA double-strand breaks. Rad60 physically interacts with, and is functionally related to, the structural maintenance of chromosomes 5 and 6 protein complex (Smc5/6). Rad60 is phosphorylated in response to hydroxyurea (HU)-induced DNA replication arrest in a Cds1(Chk2)-dependent manner. Rad60 localizes in nucleus in unchallenged cells, but becomes diffused throughout the cell in response to HU. To understand the role of Rad60 phosphorylation, we mutated the putative phosphorylation target motifs of Cds1(Chk2) and have identified two Cds1(Chk2) target residues responsible for Rad60 dispersal in response to HU. We show that the phosphorylation-defective rad60 mutation partially suppresses HU sensitivity and the elevated recombination frequency of smc6-X. Our data suggest that Rad60 phosphorylation is required to regulate homologous recombination at stalled replication forks, probably by regulating Smc5/6.

• Gluenz E, Sharma R, Carrington M, Gull K
• Functional characterization of cohesin subunit SCC1 in Trypanosoma brucei and dissection of mutant phenotypes in two life cycle stages.
• Mol Microbiol. 2008; 69: 666-80
• Display abstract

• In yeast and metazoa, structural maintenance of chromosome (SMC) complexes play key roles in chromosome segregation, architecture and DNA repair. The main function of the cohesin complex is to hold replicated sister chromatids together until segregation at anaphase, which is dependent on proteolytic cleavage of the cohesin subunit SCC1. Analysis of trypanosomatid genomes showed that the core cohesin and condensin complexes are conserved, but SMC5/6 is absent. To investigate the functional conservation of cohesin in eukaryotes distantly related to yeast and metazoa, we characterized the Trypanosoma brucei SCC1 orthologue. TbSCC1 is expressed prior to DNA synthesis at late G1, remains in the nucleus throughout S- and G2-phases of the cell cycle and disappears at anaphase. Depletion of SCC1 by RNAi or expression of a non-cleavable SCC1 resulted in karyokinesis failure. Using the dominant negative phenotype of non-cleavable SCC1 we investigated checkpoint regulation of cytokinesis in response to mitosis failure at anaphase. In the absence of chromosome segregation, procyclic trypanosomes progressed through cytokinesis to produce one nucleated and one anucleate cell (zoid). In contrast, cytokinesis was incomplete in bloodstream forms, where cleavage was initiated but cells failed to progress to abscission. Kinetoplast duplication was uninterrupted resulting in cells with multiple kinetoplasts and flagella.

• Takahashi S, Kontani K, Araki Y, Katada T
• Caf1 regulates translocation of ribonucleotide reductase by releasing nucleoplasmic Spd1-Suc22 assembly.
• Nucleic Acids Res. 2007; 35: 1187-97
• Display abstract

• Appropriate supply of deoxyribonucleotides by the ribonucleotide reductase (RNR) complex is essential for DNA replication and repair. One recent model for the RNR activation in Schizosaccharomyces pombe is translocation of the regulatory subunit Suc22 from the nucleoplasm to the cytoplasm. The RNR inhibitory protein Spd1, which retains Suc22 in the nucleoplasm, is rapidly degraded upon DNA-replication stress, resulting in release of Suc22 to form the active RNR complex in the cytoplasm. Here, we show that Caf1, a component of the Ccr4-Not complex, is responsible for resistance of the replication stress and control of the Suc22 translocation. Caf1 is required not only for the stress-induced translocation of Suc22 from nucleoplasm to cytoplasm but also for the degradation of nucleoplasmic Spd1. DNA-replication stress appears to allow Caf1 to interact with Suc22, resulting in release of the nucleoplasmic Spd1-Suc22 assembly. Taken together, these results suggest a novel function of Caf1 as a key regulator in the stress-induced RNR activation.

• Niu H et al.
• Mek1 kinase is regulated to suppress double-strand break repair between sister chromatids during budding yeast meiosis.
• Mol Cell Biol. 2007; 27: 5456-67
• Display abstract

• Mek1 is a meiosis-specific kinase in budding yeast which promotes recombination between homologous chromosomes by suppressing double-strand break (DSB) repair between sister chromatids. Previous work has shown that in the absence of the meiosis-specific recombinase gene, DMC1, cells arrest in prophase due to unrepaired DSBs and that Mek1 kinase activity is required in this situation to prevent repair of the breaks using sister chromatids. This work demonstrates that Mek1 is activated in response to DSBs by autophosphorylation of two conserved threonines, T327 and T331, in the Mek1 activation loop. Using a version of Mek1 that can be conditionally dimerized during meiosis, Mek1 function was shown to be promoted by dimerization, perhaps as a way of enabling autophosphorylation of the activation loop in trans. A putative HOP1-dependent dimerization domain within the C terminus of Mek1 has been identified. Dimerization alone, however, is insufficient for activation, as DSBs and Mek1 recruitment to the meiosis-specific chromosomal core protein Red1 are also necessary. Phosphorylation of S320 in the activation loop inhibits sister chromatid repair specifically in dmc1Delta-arrested cells. Ectopic dimerization of Mek1 bypasses the requirement for S320 phosphorylation, suggesting this phosphorylation is necessary for maintenance of Mek1 dimers during checkpoint-induced arrest.

• Hauf S, Biswas A, Langegger M, Kawashima SA, Tsukahara T, Watanabe Y
• Aurora controls sister kinetochore mono-orientation and homolog bi-orientation in meiosis-I.
• EMBO J. 2007; 26: 4475-86
• Display abstract

• Aurora-B kinases are important regulators of mitotic chromosome segregation, where they are required for the faithful bi-orientation of sister chromatids. In contrast to mitosis, sister chromatids have to be oriented toward the same spindle pole in meiosis-I, while homologous chromosomes are bi-oriented. We find that the fission yeast Aurora kinase Ark1 is required for the faithful bi-orientation of sister chromatids in mitosis and of homologous chromosomes in meiosis-I. Unexpectedly, Ark1 is also necessary for the faithful mono-orientation of sister chromatids in meiosis-I, even though the canonical mono-orientation pathway, which depends on Moa1 and Rec8, seems intact. Our data suggest that Ark1 prevents unified sister kinetochores during metaphase-I from merotelic attachment to both spindle poles and thus from being torn apart during anaphase-I, revealing a novel mechanism promoting monopolar attachment. Furthermore, our results provide an explanation for the previously enigmatic observation that fission yeast Shugoshin Sgo2, which assists in loading Aurora to centromeres, and its regulator Bub1 are required for the mono-orientation of sister chromatids in meiosis-I.

• Beaudoin J, Labbe S
• Crm1-mediated nuclear export of the Schizosaccharomyces pombe transcription factor Cuf1 during a shift from low to high copper concentrations.
• Eukaryot Cell. 2007; 6: 764-75
• Display abstract

• In this study, we examine the fate of the nuclear pool of the Schizosaccharomyces pombe transcription factor Cuf1 in response to variations in copper levels. A nuclear pool of Cuf1-green fluorescent protein (GFP) was generated by expressing a functional cuf1(+)-GFP allele in the presence of a copper chelator. We then extinguished cuf1(+)-GFP expression and tracked the changes in the localization of the nuclear pool of Cuf1-GFP in the presence of low or high copper concentrations. Treating cells with copper as well as silver ions resulted in the nuclear export of Cuf1. We identified a leucine-rich nuclear export signal (NES), (349)LAALNHISAL(358), within the C-terminal region of Cuf1. Mutations in this sequence abrogated Cuf1 export from the nucleus. Furthermore, amino acid substitutions that impair Cuf1 NES function resulted in increased target gene expression and a concomitant cellular hypersensitivity to copper. Export of the wild-type Cuf1 protein was inhibited by leptomycin B (LMB), a specific inhibitor of the nuclear export protein Crm1. We further show that cells expressing a temperature-sensitive mutation in crm1(+) exhibit increased nuclear accumulation of Cuf1 at the nonpermissive temperature. Although wild-type Cuf1 is localized in the nucleus in both conditions, we observed that the protein can still be inactivated by copper, resulting in the repression of ctr4(+) gene expression in the presence of exogenous copper. These results demonstrate that nuclear accumulation of Cuf1 per se is not sufficient to cause the unregulated expression of the copper transport genes like ctr4(+). In addition to nuclear localization, a functional Cys-rich domain or NES element in Cuf1 is required to appropriately regulate copper transport gene expression in response to changes in intracellular copper concentration.

• Memarian N et al.
• Colony size measurement of the yeast gene deletion strains for functional genomics.
• BMC Bioinformatics. 2007; 8: 117-117
• Display abstract

• BACKGROUND: Numerous functional genomics approaches have been developed to study the model organism yeast, Saccharomyces cerevisiae, with the aim of systematically understanding the biology of the cell. Some of these techniques are based on yeast growth differences under different conditions, such as those generated by gene mutations, chemicals or both. Manual inspection of the yeast colonies that are grown under different conditions is often used as a method to detect such growth differences. RESULTS: Here, we developed a computerized image analysis system called Growth Detector (GD), to automatically acquire quantitative and comparative information for yeast colony growth. GD offers great convenience and accuracy over the currently used manual growth measurement method. It distinguishes true yeast colonies in a digital image and provides an accurate coordinate oriented map of the colony areas. Some post-processing calculations are also conducted. Using GD, we successfully detected a genetic linkage between the molecular activity of the plant-derived antifungal compound berberine and gene expression components, among other cellular processes. A novel association for the yeast mek1 gene with DNA damage repair was also identified by GD and confirmed by a plasmid repair assay. The results demonstrate the usefulness of GD for yeast functional genomics research. CONCLUSION: GD offers significant improvement over the manual inspection method to detect relative yeast colony size differences. The speed and accuracy associated with GD makes it an ideal choice for large-scale functional genomics investigations.

• Miyabe I, Morishita T, Hishida T, Yonei S, Shinagawa H
• Rhp51-dependent recombination intermediates that do not generate checkpoint signal are accumulated in Schizosaccharomyces pombe rad60 and smc5/6 mutants after release from replication arrest.
• Mol Cell Biol. 2006; 26: 343-53
• Display abstract

• The Schizosaccharomyces pombe rad60 gene is essential for cell growth and is involved in repairing DNA double-strand breaks. Rad60 physically interacts with and is functionally related to the structural maintenance of chromosomes 5 and 6 (SMC5/6) protein complex. In this study, we investigated the role of Rad60 in the recovery from the arrest of DNA replication induced by hydroxyurea (HU). rad60-1 mutant cells arrested mitosis normally when treated with HU. Significantly, Rad60 function is not required during HU arrest but is required on release. However, the mutant cells underwent aberrant mitosis accompanied by irregular segregation of chromosomes, and DNA replication was not completed, as revealed by pulsed-field gel electrophoresis. The deletion of rhp51 suppressed the aberrant mitosis of rad60-1 cells and caused mitotic arrest. These results suggest that Rhp51 and Rad60 are required for the restoration of a stalled or collapsed replication fork after release from the arrest of DNA replication by HU. The rad60-1 mutant was proficient in Rhp51 focus formation after release from the HU-induced arrest of DNA replication or DNA-damaging treatment. Furthermore, the lethality of a rad60-1 rqh1Delta double mutant was suppressed by the deletion of rhp51 or rhp57. These results suggest that Rad60 is required for recombination repair at a step downstream of Rhp51. We propose that Rhp51-dependent DNA structures that cannot activate the mitotic checkpoints accumulate in rad60-1 cells.

• Lui DY, Peoples-Holst TL, Mell JC, Wu HY, Dean EW, Burgess SM
• Analysis of close stable homolog juxtaposition during meiosis in mutants of Saccharomyces cerevisiae.
• Genetics. 2006; 173: 1207-22
• Display abstract

• A unique aspect of meiosis is the segregation of homologous chromosomes at the meiosis I division. The pairing of homologous chromosomes is a critical aspect of meiotic prophase I that aids proper disjunction at anaphase I. We have used a site-specific recombination assay in Saccharomyces cerevisiae to examine allelic interaction levels during meiosis in a series of mutants defective in recombination, chromatin structure, or intracellular movement. Red1, a component of the chromosome axis, and Mnd1, a chromosome-binding protein that facilitates interhomolog interaction, are critical for achieving high levels of allelic interaction. Homologous recombination factors (Sae2, Rdh54, Rad54, Rad55, Rad51, Sgs1) aid in varying degrees in promoting allelic interactions, while the Srs2 helicase appears to play no appreciable role. Ris1 (a SWI2/SNF2 related protein) and Dot1 (a histone methyltransferase) appear to play minor roles. Surprisingly, factors involved in microtubule-mediated intracellular movement (Tub3, Dhc1, and Mlp2) appear to play no appreciable role in homolog juxtaposition, unlike their counterparts in fission yeast. Taken together, these results support the notion that meiotic recombination plays a major role in the high levels of homolog interaction observed during budding yeast meiosis.

• Pebernard S, Wohlschlegel J, McDonald WH, Yates JR 3rd, Boddy MN
• The Nse5-Nse6 dimer mediates DNA repair roles of the Smc5-Smc6 complex.
• Mol Cell Biol. 2006; 26: 1617-30
• Display abstract

• Stabilization and processing of stalled replication forks is critical for cell survival and genomic integrity. We characterize a novel DNA repair heterodimer of Nse5 and Nse6, which are nonessential nuclear proteins critical for chromosome segregation in fission yeast. The Nse5/6 dimer facilitates DNA repair as part of the Smc5-Smc6 holocomplex (Smc5/6), the basic architecture of which we define. Nse5-Nse6 [corrected] (Nse5 and Nse6) [corrected] mutants display a high level of spontaneous DNA damage and mitotic catastrophe in the absence of the master checkpoint regulator Rad3 (hATR). Nse5/6 mutants are required for the response to genotoxic agents that block the progression of replication forks, acting in a pathway that allows the tolerance of irreparable UV lesions. Interestingly, the UV sensitivity of Nse5/6 [corrected] is suppressed by concomitant deletion of the homologous recombination repair factor, Rhp51 (Rad51). Further, the viability of Nse5/6 mutants depends on Mus81 and Rqh1, factors that resolve or prevent the formation of Holliday junctions. Consistently, the UV sensitivity of cells lacking Nse5/6 can be partially suppressed by overexpressing the bacterial resolvase RusA. We propose a role for Nse5/6 mutants in suppressing recombination that results in Holliday junction formation or in Holliday junction resolution.

• De Piccoli G et al.
• Smc5-Smc6 mediate DNA double-strand-break repair by promoting sister-chromatid recombination.
• Nat Cell Biol. 2006; 8: 1032-4
• Display abstract

• DNA double-strand breaks (DSB) can arise during DNA replication, or after exposure to DNA-damaging agents, and their correct repair is fundamental for cell survival and genomic stability. Here, we show that the Smc5-Smc6 complex is recruited to DSBs de novo to support their repair by homologous recombination between sister chromatids. In addition, we demonstrate that Smc5-Smc6 is necessary to suppress gross chromosomal rearrangements. Our findings show that the Smc5-Smc6 complex is essential for genome stability as it promotes repair of DSBs by error-free sister-chromatid recombination (SCR), thereby suppressing inappropriate non-sister recombination events.

• Tsuyama T et al.
• Chromatin loading of Smc5/6 is induced by DNA replication but not by DNA double-strand breaks.
• Biochem Biophys Res Commun. 2006; 351: 935-9
• Display abstract

• Smc6, a member of the structural maintenance of chromosomes (SMC) family of proteins, forms a complex with related Smc5. Genetic analyses of yeast have demonstrated the involvement of Smc6 in DNA repair and checkpoint responses. In this study, we investigated the role of the Smc5/6 complex in higher eukaryotes by analyzing its behavior in Xenopus laevis egg extracts. Smc5/6 was loaded onto chromatin during DNA replication in a manner dependent on the initiation of DNA synthesis, and it dissociated from chromatin during mitosis. Moreover, the induction of DNA double-strand breaks following replication did not significantly affect the amount of chromatin-associated Smc6. These findings suggest that the Smc5/6 complex is regulated during the cell cycle, presumably in anticipation of DNA damage that may arise during replication.

• Aragon L
• Sumoylation: a new wrestler in the DNA repair ring.
• Proc Natl Acad Sci U S A. 2005; 102: 4661-2

• Jessberger R
• How to divorce engaged chromosomes?
• Mol Cell Biol. 2005; 25: 18-22

• Chelysheva L et al.
• AtREC8 and AtSCC3 are essential to the monopolar orientation of the kinetochores during meiosis.
• J Cell Sci. 2005; 118: 4621-32
• Display abstract

• The success of the first meiotic division relies (among other factors) on the formation of bivalents between homologous chromosomes, the monopolar orientation of the sister kinetochores at metaphase I and the maintenance of centromeric cohesion until the onset of anaphase II. The meiotic cohesin subunit, Rec8 has been reported to be one of the key players in these processes, but its precise role in kinetochore orientation is still under debate. By contrast, much less is known about the other non-SMC cohesin subunit, Scc3. We report the identification and the characterisation of AtSCC3, the sole Arabidopsis homologue of Scc3. The detection of AtSCC3 in mitotic cells, the embryo lethality of a null allele Atscc3-2, and the mitotic defects of the weak allele Atscc3-1 suggest that AtSCC3 is required for mitosis. AtSCC3 was also detected in meiotic nuclei as early as interphase, and bound to the chromosome axis from early leptotene through to anaphase I. We show here that both AtREC8 and AtSCC3 are necessary not only to maintain centromere cohesion at anaphase I, but also for the monopolar orientation of the kinetochores during the first meiotic division. We also found that AtREC8 is involved in chromosome axis formation in an AtSPO11-1-independent manner. Finally, we provide evidence for a role of AtSPO11-1 in the stability of the cohesin complex.

• Kateneva AV, Konovchenko AA, Guacci V, Dresser ME
• Recombination protein Tid1p controls resolution of cohesin-dependent linkages in meiosis in Saccharomyces cerevisiae.
• J Cell Biol. 2005; 171: 241-53
• Display abstract

• Sister chromatid cohesion and interhomologue recombination are coordinated to promote the segregation of homologous chromosomes instead of sister chromatids at the first meiotic division. During meiotic prophase in Saccharomyces cerevisiae, the meiosis-specific cohesin Rec8p localizes along chromosome axes and mediates most of the cohesion. The mitotic cohesin Mcd1p/Scc1p localizes to discrete spots along chromosome arms, and its function is not clear. In cells lacking Tid1p, which is a member of the SWI2/SNF2 family of helicase-like proteins that are involved in chromatin remodeling, Mcd1p and Rec8p persist abnormally through both meiotic divisions, and chromosome segregation fails in the majority of cells. Genetic results indicate that the primary defect in these cells is a failure to resolve Mcd1p-mediated connections. Tid1p interacts with recombination enzymes Dmc1p and Rad51p and has an established role in recombination repair. We propose that Tid1p remodels Mcd1p-mediated cohesion early in meiotic prophase to facilitate interhomologue recombination and the subsequent segregation of homologous chromosomes.

• DeWall KM, Davidson MK, Sharif WD, Wiley CA, Wahls WP
• A DNA binding motif of meiotic recombinase Rec12 (Spo11) defined by essential glycine-202, and persistence of Rec12 protein after completion of recombination.
• Gene. 2005; 356: 77-84
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• The Rec12 (Spo11) protein of the fission yeast Schizosaccharomyces pombe is a meiosis-specific ortholog of the catalytic subunit of type VI topoisomerases and is thought to catalyze double-strand DNA breaks that initiate recombination. We tested the hypothesis that the rec12-117 allele affects the choice of pathways by which recombination is resolved. DNA sequence analysis revealed a single missense mutation in the coding region (rec12-G202E). The corresponding glycine-202 residue of Rec12 protein is strictly conserved in proteins of the Rec12/Spo11/Top6A family. It maps to the base of the DNA binding pocket in the crystal structure of the archaeal ortholog, Top6A. The rec12-G202E mutants lacked crossover and non-crossover recombination, demonstrating that rec12-G202E does not affect choice of resolution pathway. Like rec12-D15 null mutants, the rec12-G202E mutants suffered chromosome segregation errors in meiosis I. The Rec12-G202E protein was as stable as wild-type Rec12, demonstrating that glycine-202 is essential for a biochemical activity of Rec12 protein, rather than for its stability. These findings suggest that Rec12 facilitates binding of the meiotic recombinase to its substrate, DNA. Interestingly, the bulk of Rec12 protein persisted until the time of anaphase I, and a portion of Rec12 protein persisted until the time of anaphase II, after which it was undetectable. This suggests that Rec12 protein has additional meiotic functions after completion of recombination in prophase, as inferred previously from genetic studies [Sharif, W.D., Glick, G.G., Davidson, M.K., Wahls, W.P., 2002. Distinct functions of S. pombe Rec12 (Spo11) protein and Rec12-dependent crossover recombination (chiasmata) in meiosis I; and a requirement for Rec12 in meiosis II. Cell Chromo. 1, 1].

• Morishita T et al.
• Role of the Schizosaccharomyces pombe F-Box DNA helicase in processing recombination intermediates.
• Mol Cell Biol. 2005; 25: 8074-83
• Display abstract

• In an effort to identify novel genes involved in recombination repair, we isolated fission yeast Schizosaccharomyces pombe mutants sensitive to methyl methanesulfonate (MMS) and a synthetic lethal with rad2. A gene that complements such mutations was isolated from the S. pombe genomic library, and subsequent analysis identified it as the fbh1 gene encoding the F-box DNA helicase, which is conserved in mammals but not conserved in Saccharomyces cerevisiae. An fbh1 deletion mutant is moderately sensitive to UV, MMS, and gamma rays. The rhp51 (RAD51 ortholog) mutation is epistatic to fbh1. fbh1 is essential for viability in stationary-phase cells and in the absence of either Srs2 or Rqh1 DNA helicase. In each case, lethality is suppressed by deletion of the recombination gene rhp57. These results suggested that fbh1 acts downstream of rhp51 and rhp57. Following UV irradiation or entry into the stationary phase, nuclear chromosomal domains of the fbh1Delta mutant shrank, and accumulation of some recombination intermediates was suggested by pulsed-field gel electrophoresis. Focus formation of Fbh1 protein was induced by treatment that damages DNA. Thus, the F-box DNA helicase appears to process toxic recombination intermediates, the formation of which is dependent on the function of Rhp51.

• Kelly JA, Williams EA, Wilce MC
• Preliminary crystallographic analysis of the Cks protein p13(suc1P90AP92A) from Schizosacharromyces pombe.
• Eur Biophys J. 2005; 34: 430-3
• Display abstract

• The p13(suc1) is the fission yeast member of the Cks (Cdc28-dependant kinase subunit) family of proteins. The Cks proteins bind to and are required for the function of cyclin-dependant kinase (Cdk) proteins during cell cycle progression in eukaryotic cells. Two conformations of Cks have been detected crystallographically; a compact monomer with the C-terminal fourth beta-strand inserted into the core of the molecule between strands 2 and 3, and a strand-exchanged dimer where the fourth beta-strand is inserted into the core of the dimer partner in an equivalent position. There is a highly conserved "hinge" region consisting of the motif PEP, N-terminal to the fourth beta-strand. In the monomer this motif constitutes a beta-turn, while in the dimeric structure it is extended, allowing strand exchange. The mutant protein p13(suc1P90AP92A), in which alanine residues replace both prolines of the turn, provides an opportunity to examine the role of the prolines in this hinge region and how they may allow for the formation of strand-exchanged dimers by Cks proteins. We have expressed and purified this mutant protein. Two millimolar p13(suc1P90AP92A) crystallised in 50 mM tris(hydroxymethyl)aminomethane pH 7.5, 30% poly(ethylene glycol) 1500. Diffraction data were collected at room temperature on an MAR345 image plate using Cu Kalpha radiation from a Rigaku RU200 rotating-anode generator source to 2.70A. The crystal has unit cell parameters a=b=75.1 A, c=34.9 A, alpha=beta=90 degrees , gamma=120 degrees. Diffraction data were indexed to the space group P6 and systematic absences 00l indicate a screw axis consistent with P6(3).

• Gillespie PJ, Hirano T
• Scc2 couples replication licensing to sister chromatid cohesion in Xenopus egg extracts.
• Curr Biol. 2004; 14: 1598-603
• Display abstract

• The cohesin complex is a central player in sister chromatid cohesion, a process that ensures the faithful segregation of chromosomes in mitosis and meiosis. Previous genetic studies in yeast show that Scc2/Mis4, a HEAT-repeat-containing protein, is required for the loading of cohesin onto chromatin. In this study, we have identified two isoforms of Scc2 in humans and Xenopus (termed Scc2A and Scc2B), which are encoded by a single gene but have different carboxyl termini created by alternative splicing. Both Scc2A and Scc2B bind to chromatin concomitant with cohesin during DNA replication in Xenopus egg extracts. Simultaneous immunodepletion of Scc2A and Scc2B from the extracts impairs the association of cohesin with chromatin, leading to severe defects in sister chromatid pairing in the subsequent mitosis. The loading of Scc2 onto chromatin is inhibited in extracts treated with geminin but not with p21(CIP1), suggesting that this step depends on replication licensing but not on the initiation of DNA replication. Upon mitotic entry, Scc2 is removed from chromatin through a mechanism that requires cdc2 but not aurora B or polo-like kinase. Our results suggest that vertebrate Scc2 couples replication licensing to sister chromatid cohesion by facilitating the loading of cohesin onto chromatin.

• Zhao H, Russell P
• DNA binding domain in the replication checkpoint protein Mrc1 of Schizosaccharomyces pombe.
• J Biol Chem. 2004; 279: 53023-7
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• The replication checkpoint is activated when replication forks are obstructed by DNA lesions or protein complexes bound to DNA or when DNA synthesis is restrained by the limited availability of deoxyribonucleotides. This checkpoint preserves genome integrity by stabilizing stalled forks and delaying the onset of mitosis. In the fission yeast Schizosaccharomyces pombe, Mrc1 is a replication checkpoint adaptor protein that allows the sensor kinase Rad3-Rad26 to activate the effector kinase Cds1. In Saccharomyces cerevisiae, Mrc1 associates with replication forks and co-precipitates with the DNA replication protein Cdc45. Whether or not Mrc1 interacts directly with DNA is unknown. Here we define a approximately 150 amino acid DNA binding domain (DBD) in the N-terminal region of S. pombe Mrc1. The DBD interacts preferentially with branched DNA structures in vitro. Deletion of the DBD or point mutations that diminish its DNA binding activity render cells sensitive to the replication inhibitor hydroxyurea. These mutations also impair the replication checkpoint arrest. The DBD has a helix-loop-helix motif that is predicted to bind DNA. This motif is conserved in the recently identified N-terminal DBD of human Claspin, a presumptive homolog of yeast Mrc1 proteins.

• Laurencon A et al.
• A large-scale screen for mutagen-sensitive loci in Drosophila.
• Genetics. 2004; 167: 217-31
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• In a screen for new DNA repair mutants, we tested 6275 Drosophila strains bearing homozygous mutagenized autosomes (obtained from C. Zuker) for hypersensitivity to methyl methanesulfonate (MMS) and nitrogen mustard (HN2). Testing of 2585 second-chromosome lines resulted in the recovery of 18 mutants, 8 of which were alleles of known genes. The remaining 10 second-chromosome mutants were solely sensitive to MMS and define 8 new mutagen-sensitive genes (mus212-mus219). Testing of 3690 third chromosomes led to the identification of 60 third-chromosome mutants, 44 of which were alleles of known genes. The remaining 16 mutants define 14 new mutagen-sensitive genes (mus314-mus327). We have initiated efforts to identify these genes at the molecular level and report here the first two identified. The HN2-sensitive mus322 mutant defines the Drosophila ortholog of the yeast snm1 gene, and the MMS- and HN2-sensitive mus301 mutant defines the Drosophila ortholog of the human HEL308 gene. We have also identified a second-chromosome mutant, mus215(ZIII-2059), that uniformly reduces the frequency of meiotic recombination to <3% of that observed in wild type and thus defines a function required for both DNA repair and meiotic recombination. At least one allele of each new gene identified in this study is available at the Bloomington Stock Center.

• Wysocka M, Rytka J, Kurlandzka A
• Saccharomyces cerevisiae CSM1 gene encoding a protein influencing chromosome segregation in meiosis I interacts with elements of the DNA replication complex.
• Exp Cell Res. 2004; 294: 592-602
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• We present the characteristics of the Csm1 (Spo86) protein of Saccharomyces cerevisiae that are important for meiotic division. The level of Csm1p does not change throughout the cell cycle, but this protein is absent in mature spores. Deletion of CSM1 causes incorrect spore formation and meiotic chromosome missegregation together with increased sensitivity of vegetative cells to benomyl and manganese. In a two-hybrid analysis with Csm1p as bait, we detected interactions with three members of the Mcm2-7 family of proteins involved in the initiation of DNA replication, and with Clf1p also implicated in replication. The Csm1p-Mcm3, Mcm5 and Mcm7p interactions were confirmed by co-immunoprecipitation. Three other interacting proteins, Mgs1p, Ulp2, and Plp2, participate in chromosome assembling and segregation, whereas the function of two others has not been established. Genetic experiments showed that the two-hybrid isolates MGS1, CLF1, MCM3, 5, 7 (CDC47), and YDL089w, when overexpressed, partially suppress the csm1Delta/csm1Delta sporulation defect. We propose that, besides its other functions, Csm1p may be involved in premeiotic DNA replication.

• Hayase A, Takagi M, Miyazaki T, Oshiumi H, Shinohara M, Shinohara A
• A protein complex containing Mei5 and Sae3 promotes the assembly of the meiosis-specific RecA homolog Dmc1.
• Cell. 2004; 119: 927-40
• Display abstract

• Meiotic recombination requires the meiosis-specific RecA homolog Dmc1 as well as the mitotic RecA homolog Rad51. Here, we show that the two meiosis-specific proteins Mei5 and Sae3 are necessary for the assembly of Dmc1, but not for Rad51, on chromosomes including the association of Dmc1 with a recombination hot spot. Mei5, Sae3, and Dmc1 form a ternary and evolutionary conserved complex that requires Rad51 for recruitment to chromosomes. Mei5, Sae3, and Dmc1 are mutually dependent for their chromosome association, and their absence prevents the disassembly of Rad51 filaments. Our results suggest that Mei5 and Sae3 are loading factors for the Dmc1 recombinase and that the Dmc1-Mei5-Sae3 complex is integrated onto Rad51 ensembles and, together with Rad51, plays both catalytic and structural roles in interhomolog recombination during meiosis.

• Khasanov FK, Salakhova AF, Chepurnaja OV, Korolev VG, Bashkirov VI
• Identification and characterization of the rlp1+, the novel Rad51 paralog in the fission yeast Schizosaccharomyces pombe.
• DNA Repair (Amst). 2004; 3: 1363-74
• Display abstract

• A new DNA repair gene from fission yeast Schizosaccharomyces pombe rlp1+ (RecA-like protein) has been identified. Rlp1 shows homology to RecA-like proteins, and is the third S. pombe Rad51 paralog besides Rhp55 and Rhp57. The new gene encodes a 363 aa protein with predicted Mr of 41,700 and has NTP-binding motif. The rlp1Delta mutant is sensitive to methyl methanesulfonate (MMS), ionizing radiation (IR), and camptothecin (CPT), although to a lesser extent than the deletion mutants of rhp55+ and rhp51+ genes. In contrast to other recombinational repair mutants, the rlp1Delta mutant does not exhibit sensitivity to UV light and mitomycin C (MMC). Mitotic recombination is moderately reduced in rlp1 mutant. Epistatic analysis of MMS and IR-sensitivity of rlp1Delta mutant indicates that rlp1+ acts in the recombinational pathway of double-strand break (DSB) repair together with rhp51+, rhp55+, and rad22+ genes. Yeast two-hybrid analysis suggests that Rlp1 may interact with Rhp57 protein. We propose that Rlp1 have an accessory role in repair of a subset of DNA damage induced by MMS and IR, and is required for the full extent of DNA recombination and cell survival under condition of a replication fork collapse.

• Perera D et al.
• TopBP1 and ATR colocalization at meiotic chromosomes: role of TopBP1/Cut5 in the meiotic recombination checkpoint.
• Mol Biol Cell. 2004; 15: 1568-79
• Display abstract

• Mammalian TopBP1 is a BRCT domain-containing protein whose function in mitotic cells is linked to replication and DNA damage checkpoint. Here, we study its possible role during meiosis in mice. TopBP1 foci are abundant during early prophase I and localize mainly to histone gamma-H2AX-positive domains, where DNA double-strand breaks (required to initiate recombination) occur. Strikingly, TopBP1 showed a pattern almost identical to that of ATR, a PI3K-like kinase involved in mitotic DNA damage checkpoint. In the synapsis-defective Fkbp6(-/-) mouse, TopBP1 heavily stains unsynapsed regions of chromosomes. We also tested whether Schizosaccharomyces pombe Cut5 (the TopBP1 homologue) plays a role in the meiotic recombination checkpoint, like spRad3, the ATR homologue. Indeed, we found that a cut5 mutation suppresses the checkpoint-dependent meiotic delay of a meiotic recombination defective mutant, indicating a direct role of the Cut5 protein in the meiotic checkpoint. Our findings suggest that ATR and TopBP1 monitor meiotic recombination and are required for activation of the meiotic recombination checkpoint.

• Bleuyard JY, Gallego ME, White CI
• Meiotic defects in the Arabidopsis rad50 mutant point to conservation of the MRX complex function in early stages of meiotic recombination.
• Chromosoma. 2004; 113: 197-203
• Display abstract

• The Rad50, Mre11 and Xrs2/Nbs1 proteins, which form the highly conserved MRX complex, perform a wide range of functions concerning the maintenance and function of DNA in eukaryotes. These include recombination, DNA repair, replication, telomere homeostasis and meiosis. Notwithstanding the attention paid to this complex, the inviability of vertebrate rad50 and mre11 mutants has led to a relative lack of information concerning the role of these proteins in meiosis in higher eukaryotes. We have previously reported that Arabidopsis atrad50 mutant plants are viable and that atrad50 mutant plants are sterile. The present study reports an analysis of the causes of this sterility and the implication of the AtRad50 protein in meiosis. Both male and female gametogenesis are defective in the Arabidopsis atrad50 mutant and cytological observation of male meiosis indicates that in the absence of the AtRad50 protein, homologous chromosomes are unable to synapse. Finally, the atrad50 mutation leads to the destruction of chromosomes during meiosis. These phenotypes support a role for the Arabidopsis MRX complex in early stages of meiotic recombination.

• Chiu A, Revenkova E, Jessberger R
• DNA interaction and dimerization of eukaryotic SMC hinge domains.
• J Biol Chem. 2004; 279: 26233-42
• Display abstract

• The eukaryotic SMC1/SMC3 heterodimer is essential for sister chromatid cohesion and acts in DNA repair and recombination. Dimerization depends on the central hinge domain present in all SMC proteins, which is flanked at each side by extended coiled-coil regions that terminate in specific globular domains. Here we report on DNA interactions of the eukaryotic, heterodimeric SMC1/SMC3 hinge regions, using the two known isoforms, SMC1alpha/SMC3 and the meiotic SMC1beta/SMC3. Both dimers bind DNA with a preference for double-stranded DNA and DNA rich in potential secondary structures. Both dimers form large protein-DNA networks and promote reannealing of complementary DNA strands. DNA binding but not dimerization depends on approximately 20 amino acids of transitional sequence into the coiled-coil region. Replacement of three highly conserved glycine residues, thought to be required for dimerization, in one of the two hinge domains still allows formation of a stable dimer, but if two hinge domains are mutated dimerization fails. Single-mutant dimers bind DNA, but hinge monomers do not. Together, we show that eukaryotic hinge dimerization does not require conserved glycines in both hinge domains, that only the transition into the coiled-coil region rather than the entire coiled-coil region is necessary for DNA binding, and that dimerization is required but not sufficient for DNA binding of the eukaryotic hinge heterodimer.

• Du LL, Moser BA, Russell P
• Homo-oligomerization is the essential function of the tandem BRCT domains in the checkpoint protein Crb2.
• J Biol Chem. 2004; 279: 38409-14
• Display abstract

• BRCT (BRCA1 C terminus) domains are frequently found as a tandem repeat in proteins involved in DNA damage responses, such as Saccharomyces cerevisiae Rad9, human 53BP1 and BRCA1. Tandem BRCT domains mediate protein-protein and protein-DNA interactions. However, the functional significance of these interactions is largely unknown. Here we report the oligomerization of Schizosaccharomyces pombe checkpoint protein Crb2 through its tandem BRCT domains. Truncated Crb2 without BRCT domains is defective in DNA damage checkpoint signaling. However, addition of either of two heterologous dimerization motifs largely restores the functions of truncated Crb2 without BRCT domains. Replacement of Crb2 BRCT domains with a dimerization motif also renders cells resistant to the dominant negative effect of overexpressing Crb2 BRCT domains. These results demonstrate that the crucial function of the tandem BRCT domains is to oligomerize Crb2.

• Morikawa H, Morishita T, Kawane S, Iwasaki H, Carr AM, Shinagawa H
• Rad62 protein functionally and physically associates with the smc5/smc6 protein complex and is required for chromosome integrity and recombination repair in fission yeast.
• Mol Cell Biol. 2004; 24: 9401-13
• Display abstract

• Smc5 and Smc6 proteins form a heterodimeric SMC (structural maintenance of chromosome) protein complex like SMC1-SMC3 cohesin and SMC2-SMC4 condensin, and they associate with non-SMC proteins Nse1 and Nse2 stably and Rad60 transiently. This multiprotein complex plays an essential role in maintaining chromosome integrity and repairing DNA double strand breaks (DSBs). This study characterizes a Schizosaccharomyces pombe mutant rad62-1, which is hypersensitive to methyl methanesulfonate (MMS) and synthetically lethal with rad2 (a feature of recombination mutants). rad62-1 is hypersensitive to UV and gamma rays, epistatic with rhp51, and defective in repair of DSBs. rad62 is essential for viability and genetically interacts with rad60, smc6, and brc1. Rad62 protein physically associates with the Smc5-6 complex. rad62-1 is synthetically lethal with mutations in the genes promoting recovery from stalled replication, such as rqh1, srs2, and mus81, and those involved in nucleotide excision repair like rad13 and rad16. These results suggest that Rad62, like Rad60, in conjunction with the Smc5-6 complex, plays an essential role in maintaining chromosome integrity and recovery from stalled replication by recombination.

• Sakai A, Hizume K, Sutani T, Takeyasu K, Yanagida M
• Condensin but not cohesin SMC heterodimer induces DNA reannealing through protein-protein assembly.
• EMBO J. 2003; 22: 2764-75
• Display abstract

• Condensin and cohesin are chromosomal protein complexes required for chromosome condensation and sister chromatid cohesion, respectively. They commonly contain the SMC (structural maintenance of chromosomes) subunits consisting of a long coiled-coil with the terminal globular domains and the central hinge. Condensin and cohesin holo-complexes contain three and two non-SMC subunits, respectively. In this study, DNA interaction with cohesin and condensin complexes purified from fission yeast was investigated. The DNA reannealing activity is strong for condensin SMC heterodimer but weak for holo-condensin, whereas no annealing activity is found for cohesin heterodimer SMC and Rad21-bound heterotrimer complexes. One set of globular domains of the same condensin SMC is essential for the DNA reannealing activity. In addition, the coiled-coil and hinge region of another SMC are needed. Atomic force microscopy discloses the molecular events of DNA reannealing. SMC assembly that occurs on reannealing DNA seems to be a necessary intermediary step. SMC is eliminated from the completed double-stranded DNA. The ability of heterodimeric SMC to reanneal DNA may be regulated in vivo possibly through the non-SMC heterotrimeric complex.

• Heyer WD, Ehmsen KT, Solinger JA
• Holliday junctions in the eukaryotic nucleus: resolution in sight?
• Trends Biochem Sci. 2003; 28: 548-57
• Display abstract

• The Holliday junction is a key recombination intermediate whose resolution generates crossovers. Interplay between recombination, repair and replication has moved the Holliday junction to the center stage of nuclear DNA metabolism. Holliday junction resolvases in the eukaryotic nucleus have long eluded identification. The endonucleases Mus81/Mms4-Eme1 and XPF-MEI-9/MUS312 are structurally related to the archaeal resolvase Hjc and were found to be involved in crossover formation in budding yeast and flies, respectively. Although these endonucleases might represent one class of eukaryotic resolvases, their substrate preference opens up the possibility that junctions other than classical Holliday junctions might contribute to crossovers. Holliday junction resolution to non-crossover products can also be achieved topologically, for example, by the action of RecQ-like DNA helicases combined with topoisomerase III.

• Siddiqui NU, Stronghill PE, Dengler RE, Hasenkampf CA, Riggs CD
• Mutations in Arabidopsis condensin genes disrupt embryogenesis, meristem organization and segregation of homologous chromosomes during meiosis.
• Development. 2003; 130: 3283-95
• Display abstract

• Proper chromatin condensation and sister chromatid resolution are essential for the maintenance of chromosomal integrity during cell division, and is in part mediated by a conserved multisubunit apparatus termed the condensin complex. The core subunits of the complex are members of the SMC2 (Structural Maintenance of Chromosomes) and SMC4 gene families. We have cloned an Arabidopsis gene, AtCAP-E1, which is a functional ortholog of the yeast SMC2 gene. A second, highly homologous SMC2 gene, AtCAPE-2, was identified by the Arabidopsis genome project. SMC2 gene expression in Arabidopsis was correlated with the mitotic activity of tissues, with high level expression observed in meristematic cells. The two genes are differentially expressed with AtCAP-E1 accounting for more than 85% of the total SMC2 transcript pool. The titan3 mutant is the result of a T-DNA insertion into AtCAP-E1, but other than subtle endosperm defects, titan3 is viable and fecund. We identified a T-DNA insertion mutant of AtCAP-E2, which showed no obvious mutant phenotype, indicating that the two genes are functionally redundant. Genetic crosses were employed to examine the consequences of reduced SMC2 levels. Both male and female gametogenesis were compromised in double mutant spores. Embryo lethality was observed for both double homozygous and AtCAP-E1(-/-), AtCAP-E2(+/-) plants; arrest occurred at or before the globular stage and was associated with altered planes of cell division in both the suspensor and the embryo. Down regulation of both genes by antisense technology, as well as in AtCAP-E1(+/-), AtCAP-E2(-/-) plants results in meristem disorganization and fasciation. Our data are consistent with the interpretation that threshold levels of SMC2 proteins are required for normal development and that AtCAP-E2 may have a higher affinity for its target than AtCAP-E1.

• Stray JE, Lindsley JE
• Biochemical analysis of the yeast condensin Smc2/4 complex: an ATPase that promotes knotting of circular DNA.
• J Biol Chem. 2003; 278: 26238-48
• Display abstract

• To better understand the contributions that the structural maintenance of chromosome proteins (SMCs) make to condensin activity, we have tested a number of biochemical, biophysical, and DNA-associated attributes of the Smc2p-Smc4p pair from budding yeast. Smc2p and Smc4p form a stable heterodimer, the "Smc2/4 complex," which upon analysis by sedimentation equilibrium appears to reversibly self-associate to form heterotetramers. Individually, neither Smc2p nor Smc4p hydrolyzes ATP; however, ATPase activity is recovered by equal molar mixing of both purified proteins. Hydrolysis activity is unaffected by the presence of DNA. Smc2/4 binds both linearized and circular plasmids, and the binding appears to be independent of adenylate nucleotide. High mole ratios of Smc2/4 to plasmid promote a geometric change in circular DNA that can be trapped as knots by type II topoisomerases but not as supercoils by a type I topoisomerase. Binding titration analyses reveal that two Smc2/4-DNA-bound states exist, one disrupted by and one resistant to salt challenge. Competition-displacement experiments show that Smc2/4-DNA-bound species formed at even high protein to DNA mole ratios remain reversible. Surprisingly, only linear and supercoiled DNA, not nicked-circular DNA, can completely displace Smc2/4 prebound to a labeled, nicked-circular DNA. To explain this geometry-dependent competition, we present two models of DNA binding by SMCs in which two DNA duplexes are captured within the inter-coil space of an Smc2/4 heterodimer. Based on these models, we propose a DNA displacement mechanism to explain how differences in geometry could affect the competitive potential of DNA.

• Merkle CJ, Karnitz LM, Henry-Sanchez JT, Chen J
• Cloning and characterization of hCTF18, hCTF8, and hDCC1. Human homologs of a Saccharomyces cerevisiae complex involved in sister chromatid cohesion establishment.
• J Biol Chem. 2003; 278: 30051-6
• Display abstract

• A growing body of evidence suggests that establishment of sister chromatid cohesion is dependent on replication fork passage over a precohesion area. In Saccharomyces cerevisiae, this process involves an alternative replication factor C (RFC) complex that contains the four small RFC subunits as well as CTF18, CTF8, and DCC1. Here, we show that an evolutionarily conserved homologous complex exists in the nucleus of human cells. We demonstrate that hCTF18, hCTF8, and hDCC1 interact with each other as well as with the p38 subunit of RFC. This alternative RFC-containing complex interacts with proliferating cell nuclear antigen but not with the Rad9/Rad1/Hus1 complex, a proliferating cell nuclear antigen-like clamp involved in the DNA damage response. hCTF18 preferentially binds chromatin during S phase, suggesting a role during replication. Our data provide evidence for the existence of an alternative RFC complex with a probable role in mammalian sister chromatid cohesion establishment.

• Watanabe Y
• [RNA binding protein Mei2, a master regulator of meiosis].
• Tanpakushitsu Kakusan Koso. 2003; 48: 398-403

• McDonald WH, Pavlova Y, Yates JR 3rd, Boddy MN
• Novel essential DNA repair proteins Nse1 and Nse2 are subunits of the fission yeast Smc5-Smc6 complex.
• J Biol Chem. 2003; 278: 45460-7
• Display abstract

• The structural maintenance of chromosomes (SMC) family of proteins play essential roles in genomic stability. SMC heterodimers are required for sister-chromatid cohesion (Cohesin: Smc1 & Smc3), chromatin condensation (Condensin: Smc2 & Smc4), and DNA repair (Smc5 & Smc6). The SMC heterodimers do not function alone and must associate with essential non-SMC subunits. To gain further insight into the essential and DNA repair roles of the Smc5-6 complex, we have purified fission yeast Smc5 and identified by mass spectrometry the co-precipitating proteins, Nse1 and Nse2. We show that both Nse1 and Nse2 interact with Smc5 in vivo, as part of the Smc5-6 complex. Nse1 and Nse2 are essential proteins and conserved from yeast to man. Loss of Nse1 and Nse2 function leads to strikingly similar terminal phenotypes to those observed for Smc5-6 inactivation. In addition, cells expressing hypomorphic alleles of Nse1 and Nse2 are, like Smc5-6 mutants, hypersensitive to DNA damage. Epistasis analysis suggests that like Smc5-6, Nse1, and Nse2 function together with Rhp51 in the homologous recombination repair of DNA double strand breaks. The results of this study strongly suggest that Nse1 and Nse2 are novel non-SMC subunits of the fission yeast Smc5-6 DNA repair complex.

• Routhier EL, Donover PS, Prendergast GC
• hob1+, the fission yeast homolog of Bin1, is dispensable for endocytosis or actin organization, but required for the response to starvation or genotoxic stress.
• Oncogene. 2003; 22: 637-48
• Display abstract

• BAR (Bin/Amphiphysin/Rvs) adapter proteins have been suggested to regulate endocytosis, actin organization, apoptosis, and transcription, but their precise roles are obscure. There are at least five mammalian genes that encode BAR adapter proteins, including the evolutionarily conserved and ubiquitously expressed Bin1/Amphiphysin-II and Bin3 genes. Bin1 holds special interest as certain splice isoforms localize to the nucleus, interact with the c-Abl and c-Myc oncoproteins, and display tumor suppressor properties. To obtain functional insights, we embarked upon a genetic analysis of the two BAR adapter proteins expressed in the fission yeast Schizosaccharomyces pombe. In a previous work, a role in actin organization and cytokinesis was identified for the Bin3 homolog hob3+. In this study, a role in stress signaling was defined for the Bin1 homolog, hob1+. Notably, hob1+ was dispensable for endocytosis, actin organization, or osmotic sensitivity. Instead, mutation of hob1+ led to slight cell elongation and faulty cell cycle arrest upon nutrient starvation. These defects were complemented by Bin1, but not by Amphiphysin-I, arguing that these genes have distinct functions despite their structural similarity. hob1 delta mutant cells were also hypersensitive to genotoxic stress. This was not related to a faulty checkpoint response, but mutation in the checkpoint gene rad3(+) further exacerbated the sensitivity of hob1 delta mutant cells. Interestingly, mutation of the cell cycle regulator wee1+ partially relieved the sensitivity defect, suggesting that hob1+ may influence the efficiency of DNA repair or checkpoint release after DNA damage. Genetic and biochemical evidence indicated that hob3+ is epistatic to hob1+ in the response to genotoxic stress. Our findings indicate that the Bin1 homolog hob1+ participates in DNA damage signaling and they suggest a novel role for BAR adapter proteins in stress response processes.

• Goshima G, Iwasaki O, Obuse C, Yanagida M
• The role of Ppe1/PP6 phosphatase for equal chromosome segregation in fission yeast kinetochore.
• EMBO J. 2003; 22: 2752-63
• Display abstract

• Mis12 is a kinetochore protein essential for equal chromosome segregation and is evolutionarily conserved from yeast to human. In this study, we report the isolation and characterization of suppressors of the mis12 mutant in fission yeast. Our results indicate that Mis12 is negatively regulated by a highly conserved protein phosphatase Ppe1 (scSit4/dmPPV/hPP6) or its bound partner Ekc1 (scSAP), and it is positively regulated by a counteracting kinase Gsk3. Mass spectrometry analysis shows that at least two sites in Mis12 are phosphorylated. This mechanism of suppression occurs at the level of localization recovery of Mis12 to the kinetochore chromatin. Consistently, Mis12 and a subpopulation of Ppe1/Ekc1 were found to behave like non-histone-type chromatin-associating proteins in the chromatin fractionation assay. Mutant analysis of Ppe1 and Ekc1 revealed that they are important for faithful chromosome segregation, as the mutants exhibited unequal chromosome segregation similar to mis12 in the presence of a low concentration of tubulin poison. Ppe1/PP6 directly or indirectly modulates kinetochore chromatin protein Mis12 to ensure progression into normal anaphase.

• Forsburg SL
• Only connect: linking meiotic DNA replication to chromosome dynamics.
• Mol Cell. 2002; 9: 703-11
• Display abstract

• The process of meiosis reduces a diploid cell to four haploid gametes and is accompanied by extensive recombination. Thus, chromosome dynamics in meiosis are significantly different than in mitotic cells. This review analyzes unique features of meiotic DNA replication and describes how it affects subsequent recombination and chromosome segregation.

• Moens PB, Kolas NK, Tarsounas M, Marcon E, Cohen PE, Spyropoulos B
• The time course and chromosomal localization of recombination-related proteins at meiosis in the mouse are compatible with models that can resolve the early DNA-DNA interactions without reciprocal recombination.
• J Cell Sci. 2002; 115: 1611-22
• Display abstract

• During mouse meiosis, the early prophase RAD51/DMC1 recombination protein sites, which are associated with the chromosome cores and which serve as markers for ongoing DNA-DNA interactions, are in ten-fold excess of the eventual reciprocal recombinant events. Most, if not all, of these early interactions are eliminated as prophase progresses. The manner in which these sites are eliminated is the focus of this investigation. We report that these sites acquire replication protein A, RPA and the Escherichia coli MUTS homologue, MSH4p, and somewhat later the Bloom helicase, BLM, while simultaneously losing the RAD51/DMC1 component. Eventually the RPA component is also lost and BLM sites remain. At that time, the MUTL homologue, MLH1p, which is essential for reciprocal recombination in the mouse, appears in numbers and locations that correspond to the distribution of reciprocal recombination events. However, the MLH1 foci do not appear to coincide with the remaining BLM sites. The MLH1p is specifically localized to electron-microscope-defined recombination nodules. We consider the possibility that the homology-search RAD51/DMC1 complexes are involved in homologous chromosome synapsis but that most of these early DNA-DNA interactions are later resolved by the anti-recombination RPA/MSH4/BLM-topoisomerase complex, thereby preventing the formation of superfluous reciprocal recombinant events.

• Peggie MW, MacKelvie SH, Bloecher A, Knatko EV, Tatchell K, Stark MJ
• Essential functions of Sds22p in chromosome stability and nuclear localization of PP1.
• J Cell Sci. 2002; 115: 195-206
• Display abstract

• Sds22p is a conserved, leucine-rich repeat protein that interacts with the catalytic subunit of protein phosphatase 1 (PP1(C)) and which has been proposed to regulate one or more functions of PP1(C) during mitosis. Here we show that Saccharomyces cerevisiae Sds22p is a largely nuclear protein, most of which is present as a sTable 1:1 complex with yeast PP1(C) (Glc7p). Temperature-sensitive (Ts(-)) S. cerevisiae sds22 mutants show profound chromosome instability at elevated growth temperatures but do not confer a cell cycle stage-specific arrest. In the sds22-6 Ts(-) mutant, nuclear Glc7p is both reduced in level and aberrantly localized at 37 degrees C and the interaction between Glc7p and Sds22p in vitro is reduced at higher temperatures, consistent with the in vivo Ts(-) growth defect. Like some glc7 mutations, sds22-6 can suppress the Ts(-) growth defect associated with ipl1-2, a loss of function mutation in a protein kinase that is known to work in opposition to PP1 on at least two nuclear substrates. This, together with reciprocal genetic interactions between GLC7 and SDS22, suggests that Sds22p functions positively with Glc7p to promote dephosphorylation of nuclear substrates required for faithful transmission of chromosomes during mitosis, and this role is at least partly mediated by effects of Sds22p on the nuclear distribution of Glc7p

• De DN
• Protein constitution of the chromosome axis.
• Chromosoma. 2002; 111: 69-79
• Display abstract

• Diverse studies indicate that certain nonhistone proteins may be responsible for maintaining the integrity of the eukaryotic chromosomes. In view of their dispersal during nuclear division and their acknowledged role in gene expression, none of the nuclear matrix proteins can be regarded as integral proteins of the chromosome. Topoisomerase II, structural maintenance of chromosome (SMC) proteins and high-mobility-group (HMG) proteins exhibit characteristic enzymatic and mechanochemical roles as well as transitory association with chromosomes. Hence they may be regarded as accessory chromosome proteins. In contrast, certain high molecular weight synaptonemal complex proteins (SCPs) exhibit chemical and physical properties that suggest a structural role as the chromosome axis. It is proposed in an experimentally testable model that, in the minimal structure of a eukaryotic chromosome, the terminals of the loops of DNA-histone solenoids are constitutively affixed to chromosome axis proteins that have elastic properties.

• Elder RT, Song XQ, Chen M, Hopkins KM, Lieberman HB, Zhao Y
• Involvement of rhp23, a Schizosaccharomyces pombe homolog of the human HHR23A and Saccharomyces cerevisiae RAD23 nucleotide excision repair genes, in cell cycle control and protein ubiquitination.
• Nucleic Acids Res. 2002; 30: 581-91
• Display abstract

• A functional homolog (rhp23) of human HHR23A and Saccharomyces cerevisiae RAD23 was cloned from the fission yeast Schizosaccharomyces pombe and characterized. Consistent with the role of Rad23 homologs in nucleotide excision repair, rhp23 mutant cells are moderately sensitive to UV light but demonstrate wild-type resistance to gamma-rays and hydroxyurea. Expression of the rhp23, RAD23 or HHR23A cDNA restores UV resistance to the mutant, indicating that rhp23 is a functional homolog of the human and S.cerevisiae genes. The rhp23::ura4 mutation also causes a delay in the G2 phase of the cell cycle which is corrected when rhp23, RAD23 or HHR23A cDNA is expressed. Rhp23 is present throughout the cell but is located predominantly in the nucleus, and the nuclear levels of Rhp23 decrease around the time of S phase in the cell cycle. Rhp23 is ubiquitinated at low levels, but overexpression of the rhp23 cDNA induces a large increase in ubiquitination of other proteins. Consistent with a role in protein ubiquitination, Rhp23 binds ubiquitin, as determined by two-hybrid analysis. Thus, the rhp23 gene plays a role not only in nucleotide excision repair but also in cell cycle regulation and the ubiquitination pathways.

• Ohi MD, Link AJ, Ren L, Jennings JL, McDonald WH, Gould KL
• Proteomics analysis reveals stable multiprotein complexes in both fission and budding yeasts containing Myb-related Cdc5p/Cef1p, novel pre-mRNA splicing factors, and snRNAs.
• Mol Cell Biol. 2002; 22: 2011-24
• Display abstract

• Schizosaccharomyces pombe Cdc5p and its Saccharomyces cerevisiae ortholog, Cef1p, are essential Myb-related proteins implicated in pre-mRNA splicing and contained within large multiprotein complexes. Here we describe the tandem affinity purification (TAP) of Cdc5p- and Cef1p-associated complexes. Using transmission electron microscopy, we show that the purified Cdc5p complex is a discrete structure. The components of the S. pombe Cdc5p/S. cerevisiae Cef1p complexes (termed Cwfs or Cwcs, respectively) were identified using direct analysis of large protein complex (DALPC) mass spectrometry (A. J. Link et al., Nat. Biotechnol. 17:676-682, 1999). At least 26 proteins were detected in the Cdc5p/Cef1p complexes. Comparison of the polypeptides identified by S. pombe Cdc5p purification with those identified by S. cerevisiae Cef1p purification indicates that these two yeast complexes are nearly identical in composition. The majority of S. pombe Cwf proteins and S. cerevisiae Cwc proteins are known pre-mRNA splicing factors including core Sm and U2 and U5 snRNP components. In addition, the complex contains the U2, U5, and U6 snRNAs. Previously uncharacterized proteins were also identified, and we provide evidence that several of these novel factors are involved in pre-mRNA splicing. Our data represent the first comprehensive analysis of CDC5-associated proteins in yeasts, describe a discrete highly conserved complex containing novel pre-mRNA splicing factors, and demonstrate the power of DALPC for identification of components in multiprotein complexes.

• Tougan T, Chiba Y, Kakihara Y, Hirata A, Nojima H
• Meu10 is required for spore wall maturation in Schizosaccharomyces pombe.
• Genes Cells. 2002; 7: 217-31
• Display abstract

• BACKGROUND: Many genes are meiosis and/or sporulation-specifically transcribed during this process. Isolation and analysis of these genes might help us to understand how meiosis and sporulation are regulated. For this purpose, we have isolated a large number of cDNA clones from Schizosaccharomyces pombe whose expression is up-regulated during meiosis. RESULTS: We have isolated meu10+ gene, which encodes 416 amino acids and bears homology to SPS2 of Saccharomyces cerevisiae. A strain whose meu10+ gene has been deleted forms no viable spores. Thin-section electron micrographs showed that the meu10Delta strain has abnormally formed spore walls, and then they disrupt, allowing cytoplasmic material to escape. The Meu10-GFP fusion protein is localized to the spore periphery, thereafter returned to the cytoplasm after sporulation. Meu10-GFP localization to the spore wall was almost normal in the bgs2Delta or chs1Delta mutants that lack 1,3-beta-glucan or chitin, respectively. In contrast, 1,3-beta-glucan is abnormally localized in meu10Delta cells. Meu10 has an N-terminal domain with homology to the mammalian insulin receptor and a C-terminal domain with a transmembrane motif. Mutants whose N-terminal or C-terminal domain was truncated were severely defective for sporulation. CONCLUSIONS: Meu10 is a spore wall component and plays a pivotal role in the formation of the mature spore wall structure.

• Aono N, Sutani T, Tomonaga T, Mochida S, Yanagida M
• Cnd2 has dual roles in mitotic condensation and interphase.
• Nature. 2002; 417: 197-202
• Display abstract

• Chromosome condensation requires condensin, which comprises five subunits. Two of these subunits--both being structural maintenance of chromosome (SMC) proteins-are coiled-coils with globular terminal domains that interact with ATP and DNA. The remaining three, non-SMC subunits also have essential, albeit undefined, roles in condensation. Here we report that Cnd2 (ref. 6), a non-SMC subunit of fission yeast similar to Drosophila Barren and the budding yeast protein Brn1 (refs 8, 9), is required for both interphase and mitotic condensation. In cnd2-1 mutants, ultraviolet-induced DNA damage is not repaired, and cells arrested by hydroxyurea do not recover. A definitive defect of interphase is abolishment of Cds1 (a checkpoint kinase) activation in the presence of hydroxyurea in both cnd2-1 mutant cells and in cells where other condensin subunits have been genetically disrupted. In the absence of hydroxyurea, a G2 checkpoint delay occurred in cnd2-1 mutants in a manner dependent on Cds1 and ATM-like Rad3, but not Chk1 (refs 10-13), before the mitotic condensation defect. Furthermore, cnd2-1 was synthetic-lethal with mutations of excision repair, RecQ helicase and DNA replication enzymes. These interphase and mitotic defects provide insight into the mechanistic role of non-SMC subunits that interact with the globular SMC domains in the heteropentameric holocomplex.

• van den Bosch M, Zonneveld JB, Vreeken K, de Vries FA, Lohman PH, Pastink A
• Differential expression and requirements for Schizosaccharomyces pombe RAD52 homologs in DNA repair and recombination.
• Nucleic Acids Res. 2002; 30: 1316-24
• Display abstract

• In fission yeast two RAD52 homologs have been identified, rad22A(+) and rad22B(+). Two-hybrid experiments and GST pull-down assays revealed physical interaction between Rad22A and Rad22B, which is dependent on the N-terminal regions. Interaction with Rhp51 is dependent on the C-terminal parts of either protein. Both Rad22A and Rad22B also interact with RPA. The expression of rad22B(+) in mitotically dividing cells is very low in comparison with rad22A(+) but is strongly enhanced after induction of meiosis, in contrast to rad22A(+). Rad22B mutant cells are not hypersensitive to DNA-damaging agents (X-rays, UV and cisplatin) and display normal levels of recombination. In these respects the Schizosaccharomyces pombe rad22B mutant resembles the weak phenotype of vertebrate cells deficient for RAD52. Mutation of rad22A(+) leads to severe sensitivity to DNA-damaging agents and to defects in recombination. In a rad22Arad22B double mutant a further increase in sensitivity to DNA-damaging agents and additional mitotic recombination defects were observed. The data presented here indicate that Rad22A and Rad22B have overlapping roles in repair and recombination, although specialized functions for each protein cannot be excluded.

• Nakamura T, Nakamura-Kubo M, Nakamura T, Shimoda C
• Novel fission yeast Cdc7-Dbf4-like kinase complex required for the initiation and progression of meiotic second division.
• Mol Cell Biol. 2002; 22: 309-20
• Display abstract

• Cdc7, a conserved serine/threonine protein kinase, controls initiation of DNA replication. A regulatory subunit, Dbf4, stimulates the kinase activity of Cdc7 and recruits it to the replication origins. Schizosaccharomyces pombe has a homologous kinase complex, composed of Hsk1 and Dfp1/Him1. Here, we report a novel protein kinase of S. pombe, Spo4, which shares common structural features with the Cdc7 kinases. In spite of the structural similarities, Spo4 is dispensable for mitotic growth and premeiotic DNA replication. Intriguingly, spo4 null mutants are defective in initiation and progression of the second meiotic division. Spindles for meiosis II are often fragmented. Spo4 kinase activity is markedly enhanced when the enzyme is associated with its regulatory subunit, Spo6, a Dbf4-like protein. Expression of Spo4 is specifically induced during meiosis. Spo4 is preferentially present in nuclei, but this nuclear localization does not require Spo6. These results suggest that Spo4 is a Cdc7 kinase whose primary role is in meiosis, not in DNA replication. This is the first report of an organism which has two Cdc7-related kinase complexes with different biological functions.

• Barker PA, Salehi A
• The MAGE proteins: emerging roles in cell cycle progression, apoptosis, and neurogenetic disease.
• J Neurosci Res. 2002; 67: 705-12
• Display abstract

• Since the identification of the first MAGE gene in 1991, the MAGE family has expanded dramatically, and over 25 MAGE genes have now been identified in humans. The focus of studies on the MAGE proteins has been their potential for cancer immunotherapy, as a result of the finding that peptides derived from MAGE gene products are bound by major histocompatibility complexes and presented on the cell surface of cancer cells. However, the normal physiological role of MAGE proteins has remained a mystery. Recent studies are now beginning to provide insights into MAGE gene function. Necdin acts as a cell cycle regulatory protein and plays a key role in the pathogenesis of Prader-Willi syndrome, a neurogenetic disorder. MAGE-D1, identified as a binding partner for the p75 neurotrophin receptor, the apoptosis inhibitory protein XIAP, and Dlx/MSX homeodomain proteins, blocks cell cycle progression and enhances apoptosis. This review provides an overview of the human MAGE genes and proteins, summarizes recent findings on their cellular roles, and provides a baseline for future studies on this intriguing gene family.

• McDonald WH, Yates JR 3rd
• Shotgun proteomics and biomarker discovery.
• Dis Markers. 2002; 18: 99-105
• Display abstract

• Coupling large-scale sequencing projects with the amino acid sequence information that can be gleaned from tandem mass spectrometry (MS/MS) has made it much easier to analyze complex mixtures of proteins. The limits of this "shotgun" approach, in which the protein mixture is proteolytically digested before separation, can be further expanded by separating the resulting mixture of peptides prior to MS/MS analysis. Both single dimensional high pressure liquid chromatography (LC) and multidimensional LC (LC/LC) can be directly interfaced with the mass spectrometer to allow for automated collection of tremendous quantities of data. While there is no single technique that addresses all proteomic challenges, the shotgun approaches, especially LC/LC-MS/MS-based techniques such as MudPIT (multidimensional protein identification technology), show advantages over gel-based techniques in speed, sensitivity, scope of analysis, and dynamic range. Advances in the ability to quantitate differences between samples and to detect for an array of post-translational modifications allow for the discovery of classes of protein biomarkers that were previously unassailable.

• Prieto I et al.
• STAG2 and Rad21 mammalian mitotic cohesins are implicated in meiosis.
• EMBO Rep. 2002; 3: 543-50
• Display abstract

• STAG/SA proteins are specific cohesin complex subunits that maintain sister chromatid cohesion in mitosis and meiosis. Two members of this family, STAG1/SA1 and STAG2/SA2,double dagger are classified as mitotic cohesins, as they are found in human somatic cells and in Xenopus laevis as components of the cohesin(SA1) and cohesin(SA2) complexes, in which the shared subunits are Rad21/SCC1, SMC1 and SMC3 proteins. A recently reported third family member, STAG3, is germinal cell-specific and is a subunit of the meiotic cohesin complex. To date, the meiosis-specific cohesin complex has been considered to be responsible for sister chromatid cohesion during meiosis. We studied replacement of the mitotic by the meiotic cohesin complex during mouse germinal cell maturation, and we show that mammalian STAG2 and Rad21 are also involved in several meiosis stages. Immunofluorescence results suggest that a cohesin complex containing Rad21 and STAG2 cooperates with a STAG3-specific complex to maintain sister chromatid cohesion during the diplotene stage of meiosis.

• Ohi MD, Gould KL
• Characterization of interactions among the Cef1p-Prp19p-associated splicing complex.
• RNA. 2002; 8: 798-815
• Display abstract

• Schizosaccharomyces pombe (Sp) Cdc5p and its Saccharomyces cerevisiae (Sc) ortholog, Cef1p, are essential components of the spliceosome. In S. cerevisiae, a subcomplex of the spliceosome that includes Cef1p can be isolated on its own; this has been termed the nineteen complex (Ntc) because it contains Prp19p. Components of the Ntc include Cef1p, Snt309p, Syf2p/Ntc31p, Ntc30p/lsy1p, Ntc20p and at least six unidentified proteins. We recently identified approximately 30 proteins that copurified with Cdc5p and Cef1p. Previously unidentified S. pombe proteins in this purification were called Cwfs for complexed with five and novel S. cerevisiae proteins were called Cwcs for complexed with Cef1p. Using these proteomics data coupled with available information regarding Ntc composition, we have investigated protein identities and interactions among Ntc components. Our data indicate that Cwc2p, Prp46p, Clf1p, and Syf1p most likely represent Ntc40p, Ntc50p, Ntc77p, and Ntc90p, respectively. We show that Sc Cwc2p interacts with Prp19p and is involved in pre-mRNA splicing. Sp cwf2+, the homolog of Sc CWC2, is allelic with the previously identified Sp prp3+. We present evidence that Sp Cwf7p, an essential protein with obvious homologs in many eukaryotes but not S. cerevisiae, is a functional counterpart of Sc Snt309p and binds Sp Cwf8p (a homolog of Sc Prp19p). Further, our data indicate that a mutation in the U-box of Prp19p disrupts these numerous protein interactions causing Cef1p degradation and Ntc instability.

• Kim ST, Xu B, Kastan MB
• Involvement of the cohesin protein, Smc1, in Atm-dependent and independent responses to DNA damage.
• Genes Dev. 2002; 16: 560-70
• Display abstract

• Structural maintenance of chromosomes (SMC) proteins play important roles in sister chromatid cohesion, chromosome condensation, sex-chromosome dosage compensation, and DNA recombination and repair. Protein complexes containing heterodimers of the Smc1 and Smc3 proteins have been implicated specifically in both sister chromatid cohesion and DNA recombination. Here, we show that the protein kinase, Atm, which belongs to a family of phosphatidylinositol 3-kinases that regulate cell cycle checkpoints and DNA recombination and repair, phosphorylates Smc1 protein after ionizing irradiation. Atm phosphorylates Smc1 on serines 957 and 966 in vitro and in vivo, and expression of an Smc1 protein mutated at these phosphorylation sites abrogates the ionizing irradiation-induced S phase cell cycle checkpoint. Optimal phosphorylation of these sites in Smc1 after ionizing irradiation also requires the presence of the Atm substrates Nbs1 and Brca1. These same sites in Smc1 are phosphorylated after treatment with UV irradiation or hydroxyurea in an Atm-independent manner, thus demonstrating that another kinase must be involved in responses to these cellular stresses. Yeast containing hypomorphic mutations in SMC1 and human cells overexpressing Smc1 mutated at both of these phosphorylation sites exhibit decreased survival following ionizing irradiation. These results demonstrate that Smc1 participates in cellular responses to DNA damage and link Smc1 to the Atm signal transduction pathway.

• Kim WJ, Park EJ, Lee H, Seong RH, Park SD
• Physical interaction between recombinational proteins Rhp51 and Rad22 in Schizosaccharomyces pombe.
• J Biol Chem. 2002; 277: 30264-70
• Display abstract

• In eukaryotes, Rad51 and Rad52 are two key components of homologous recombination and recombinational repair. These two proteins interact with each other. Here we investigated the role of interaction between Rhp51 and Rad22, the fission yeast homologs of Rad51 and Rad52, respectively, on the function of each protein. We identified a direct association between the two proteins and their self-interactions both in vivo and in vitro. We also determined the binding domains of each protein that mediate these interactions. To characterize the role of Rhp51-Rad22 interaction, we used random mutagenesis to identify the mutants Rhp51 and Rad22, which cannot interact each other. Interestingly, we found that mutant Rhp51 protein, which cannot interact with either Rad22 or Rti1 (G282D), lost its DNA repair ability. In contrast, mutant Rad22 proteins, which cannot specifically bind to Rhp51 (S379L and P381L), maintained their DNA repair ability. These results suggest that the interaction between Rhp51 and Rad22 is crucial for the recombinational repair function of Rhp51. However, the significance of this interaction on the function of Rad22 remains to be characterized further.

• Fabre F, Chan A, Heyer WD, Gangloff S
• Alternate pathways involving Sgs1/Top3, Mus81/ Mms4, and Srs2 prevent formation of toxic recombination intermediates from single-stranded gaps created by DNA replication.
• Proc Natl Acad Sci U S A. 2002; 99: 16887-92
• Display abstract

• Toxic recombination events are detected in vegetative Saccharomyces cerevisiae cells through negative growth interactions between certain combinations of mutations. For example, mutations affecting both the Srs2 and Sgs1 helicases result in extremely poor growth, a phenotype suppressed by mutations in genes that govern early stages of recombination. Here, we identify a similar interaction involving double mutations affecting Sgs1 or Top3 and Mus81 or Mms4. We also find that the primary DNA structures that initiate these toxic recombination events cannot be double-strand breaks and thus are likely to be single-stranded DNA. We interpret our results in the context of the idea that replication stalling leaves single-stranded DNA, which can then be processed by two competing mechanisms: recombination and nonrecombination gap-filling. Functions involved in preventing toxic recombination would either avoid replicative defects or act on recombination intermediates. Our results suggest that Srs2 channels recombination intermediates back into the gap-filling route, whereas Sgs1Top3 and Mus81Mms4 are involved in recombination andor in replication to allow replication restart.

• Davis L, Smith GR
• Meiotic recombination and chromosome segregation in Schizosaccharomyces pombe.
• Proc Natl Acad Sci U S A. 2001; 98: 8395-402
• Display abstract

• In most organisms homologous recombination is vital for the proper segregation of chromosomes during meiosis, the formation of haploid sex cells from diploid precursors. This review compares meiotic recombination and chromosome segregation in the fission yeast Schizosaccharomyces pombe and the distantly related budding yeast Saccharomyces cerevisiae, two especially tractable microorganisms. Certain features, such as the occurrence of DNA breaks associated with recombination, appear similar, suggesting that these features may be common in eukaryotes. Other features, such as the role of these breaks and the ability of chromosomes to segregate faithfully in the absence of recombination, appear different, suggesting multiple solutions to the problems faced in meiosis.

• Taylor EM, Moghraby JS, Lees JH, Smit B, Moens PB, Lehmann AR
• Characterization of a novel human SMC heterodimer homologous to the Schizosaccharomyces pombe Rad18/Spr18 complex.
• Mol Biol Cell. 2001; 12: 1583-94
• Display abstract

• The structural maintenance of chromosomes (SMC) protein encoded by the fission yeast rad18 gene is involved in several DNA repair processes and has an essential function in DNA replication and mitotic control. It has a heterodimeric partner SMC protein, Spr18, with which it forms the core of a multiprotein complex. We have now isolated the human orthologues of rad18 and spr18 and designated them hSMC6 and hSMC5. Both proteins are about 1100 amino acids in length and are 27-28% identical to their fission yeast orthologues, with much greater identity within their N- and C-terminal globular domains. The hSMC6 and hSMC5 proteins interact to form a tight complex analogous to the yeast Rad18/Spr18 heterodimer. In proliferating human cells the proteins are bound to both chromatin and the nucleoskeleton. In addition, we have detected a phosphorylated form of hSMC6 that localizes to interchromatin granule clusters. Both the total level of hSMC6 and its phosphorylated form remain constant through the cell cycle. Both hSMC5 and hSMC6 proteins are expressed at extremely high levels in the testis and associate with the sex chromosomes in the late stages of meiotic prophase, suggesting a possible role for these proteins in meiosis.

• de los Santos T, Loidl J, Larkin B, Hollingsworth NM
• A role for MMS4 in the processing of recombination intermediates during meiosis in Saccharomyces cerevisiae.
• Genetics. 2001; 159: 1511-25
• Display abstract

• The MMS4 gene of Saccharomyces cerevisiae was originally identified due to its sensitivity to MMS in vegetative cells. Subsequent studies have confirmed a role for MMS4 in DNA metabolism of vegetative cells. In addition, mms4 diploids were observed to sporulate poorly. This work demonstrates that the mms4 sporulation defect is due to triggering of the meiotic recombination checkpoint. Genetic, physical, and cytological analyses suggest that MMS4 functions after the single end invasion step of meiotic recombination. In spo13 diploids, red1, but not mek1, is epistatic to mms4 for sporulation and spore viability, suggesting that MMS4 may be required only when homologs are capable of undergoing synapsis. MMS4 and MUS81 are in the same epistasis group for spore viability, consistent with biochemical data that show that the two proteins function in a complex. In contrast, MMS4 functions independently of MSH5 in the production of viable spores. We propose that MMS4 is required for the processing of specific recombination intermediates during meiosis.

• Boddy MN, Gaillard PH, McDonald WH, Shanahan P, Yates JR 3rd, Russell P
• Mus81-Eme1 are essential components of a Holliday junction resolvase.
• Cell. 2001; 107: 537-48
• Display abstract

• Mus81, a fission yeast protein related to the XPF subunit of ERCC1-XPF nucleotide excision repair endonuclease, is essential for meiosis and important for coping with stalled replication forks. These processes require resolution of X-shaped DNA structures known as Holliday junctions. We report that Mus81 and an associated protein Eme1 are components of an endonuclease that resolves Holliday junctions into linear duplex products. Mus81 and Eme1 are required during meiosis at a late step of meiotic recombination. The mus81 meiotic defect is rescued by expression of a bacterial Holliday junction resolvase. These findings constitute strong evidence that Mus81 and Eme1 are subunits of a nuclear Holliday junction resolvase.

• Pelttari J et al.
• A meiotic chromosomal core consisting of cohesin complex proteins recruits DNA recombination proteins and promotes synapsis in the absence of an axial element in mammalian meiotic cells.
• Mol Cell Biol. 2001; 21: 5667-77
• Display abstract

• The behavior of meiotic chromosomes differs in several respects from that of their mitotic counterparts, resulting in the generation of genetically distinct haploid cells. This has been attributed in part to a meiosis-specific chromatin-associated protein structure, the synaptonemal complex. This complex consist of two parallel axial elements, each one associated with a pair of sister chromatids, and a transverse filament located between the synapsed homologous chromosomes. Recently, a different protein structure, the cohesin complex, was shown to be associated with meiotic chromosomes and to be required for chromosome segregation. To explore the functions of the two different protein structures, the synaptonemal complex and the cohesin complex, in mammalian male meiotic cells, we have analyzed how absence of the axial element affects early meiotic chromosome behavior. We find that the synaptonemal complex protein 3 (SCP3) is a main determinant of axial-element assembly and is required for attachment of this structure to meiotic chromosomes, whereas SCP2 helps shape the in vivo structure of the axial element. We also show that formation of a cohesin-containing chromosomal core in meiotic nuclei does not require SCP3 or SCP2. Our results also suggest that the cohesin core recruits recombination proteins and promotes synapsis between homologous chromosomes in the absence of an axial element. A model for early meiotic chromosome pairing and synapsis is proposed.

• Sjogren C, Nasmyth K
• Sister chromatid cohesion is required for postreplicative double-strand break repair in Saccharomyces cerevisiae.
• Curr Biol. 2001; 11: 991-5
• Display abstract

• The repair of DNA double-strand breaks by recombination requires the presence of an undamaged copy that is used as a template during the repair process. Because cells acquire resistance to gamma irradiation during DNA replication and because sister chromatids are the preferred partner for double-strand break repair in mitotic diploid yeast cells, it has long been suspected that cohesion between sister chromatids might be crucial for efficient repair. This hypothesis is consistent with the sensitivity to gamma irradiation of mutants defective in the cohesin complex that holds sister chromatids together from DNA replication until the onset of anaphase (reviewed in) . It is also in accordance with the finding that surveillance mechanisms (checkpoints) that sense DNA damage arrest cell cycle progression in yeast by causing stabilization of the securin Pds1, thereby blocking sister chromatid separation. The hypersensitivity to irradiation of cohesin mutants could, however, be due to a more direct involvement of the cohesin complex in the process of DNA repair. We show here that passage through S phase in the presence of cohesin, and not cohesin per se, is essential for efficient double-strand break repair during G2 in yeast. Proteins needed to load cohesin onto chromosomes (Scc2) and to generate cohesion during S phase (Eco1) are also shown to be required for repair. Our results confirm what has long been suspected but never proven, that cohesion between sister chromatids is essential for efficient double-strand break repair in mitotic cells.

• Nabeshima K, Kakihara Y, Hiraoka Y, Nojima H
• A novel meiosis-specific protein of fission yeast, Meu13p, promotes homologous pairing independently of homologous recombination.
• EMBO J. 2001; 20: 3871-81
• Display abstract

• Meiotic homologous pairing is crucial to proper homologous recombination, which secures subsequent reductional chromosome segregation. We have identified a novel meiosis-specific protein of fission yeast Schizosaccharomyces pombe, Meu13p, to be a molecule that is required for proper homologous pairing and recombination. Rec12p (homologue of Saccharomyces cerevisiae Spo11p), which is essential for the initiation of meiotic recombination, is also shown for the first time to participate in the pairing process of S.pombe. Meu13p, however, contributes to pairing through a recombination-independent mechanism, as disruption of the meu13(+) gene reduces pairing whether the rec12(+) gene is deleted or not. We also demonstrate a dynamic nature of homologous pairing in living meiotic cells, which is markedly affected by meu13 deletion. Meu13p is not required for telomere clustering and the nuclear movement process, which are well known requirements for efficient pairing in S.pombe. Based on these results, together with the localization of Meu13p on meiotic chromatin, we propose that Meu13p directly promotes proper homologous pairing and recombination.

• Zenvirth D, Simchen G
• Meiotic double-strand breaks in Schizosaccharomyces pombe.
• Curr Genet. 2000; 38: 33-8
• Display abstract

• Meiotic DNA double-strand breaks (DSBs) are associated with recombination hot spots in the yeast Saccharomyces cerevisiae and are believed to initiate the process of recombination. Until now, meiosis-induced breaks have not been shown to occur regularly in other organisms. Here we show, by pulsed-field gel electrophoresis of DNA, that meiotic DSBs occur transiently in all three chromosomes of the fission yeast Schizosaccharomyces pombe. In a repair defective mutant, carrying a mutation in the RecA homolog gene rhp51, meiotic DSBs accumulate. In contrast to expectation from the genetic map of S. pombe, however, many chromosomal DNA molecules remain unbroken during meiosis.

• Boudrez A et al.
• NIPP1-mediated interaction of protein phosphatase-1 with CDC5L, a regulator of pre-mRNA splicing and mitotic entry.
• J Biol Chem. 2000; 275: 25411-7
• Display abstract

• NIPP1 is a regulatory subunit of a species of protein phosphatase-1 (PP1) that co-localizes with splicing factors in nuclear speckles. We report that the N-terminal third of NIPP1 largely consists of a Forkhead-associated (FHA) protein interaction domain, a known phosphopeptide interaction module. A yeast two-hybrid screening revealed an interaction between this domain and a human homolog (CDC5L) of the fission yeast protein cdc5, which is required for G(2)/M progression and pre-mRNA splicing. CDC5L and NIPP1 co-localized in nuclear speckles in COS-1 cells. Furthermore, an interaction between CDC5L, NIPP1, and PP1 in rat liver nuclear extracts could be demonstrated by co-immunoprecipitation and/or co-purification experiments. The binding of the FHA domain of NIPP1 to CDC5L was dependent on the phosphorylation of CDC5L, e.g. by cyclin E-Cdk2. When expressed in COS-1 or HeLa cells, the FHA domain of NIPP1 did not affect the number of cells in the G(2)/M transition. However, the FHA domain blocked beta-globin pre-mRNA splicing in nuclear extracts. A mutation in the FHA domain that abolished its interaction with CDC5L also canceled its anti-splicing effects. We suggest that NIPP1 either targets CDC5L or an associated protein for dephosphorylation by PP1 or serves as an anchor for both PP1 and CDC5L.

• Joazeiro CA, Weissman AM
• RING finger proteins: mediators of ubiquitin ligase activity.
• Cell. 2000; 102: 549-52

• Schmiesing JA, Gregson HC, Zhou S, Yokomori K
• A human condensin complex containing hCAP-C-hCAP-E and CNAP1, a homolog of Xenopus XCAP-D2, colocalizes with phosphorylated histone H3 during the early stage of mitotic chromosome condensation.
• Mol Cell Biol. 2000; 20: 6996-7006
• Display abstract

• Structural maintenance of chromosomes (SMC) family proteins play critical roles in structural changes of chromosomes. Previously, we identified two human SMC family proteins, hCAP-C and hCAP-E, which form a heterodimeric complex (hCAP-C-hCAP-E) in the cell. Based on the sequence conservation and mitotic chromosome localization, hCAP-C-hCAP-E was determined to be the human ortholog of the Xenopus SMC complex, XCAP-C-XCAP-E. XCAP-C-XCAP-E is a component of the multiprotein complex termed condensin, required for mitotic chromosome condensation in vitro. However, presence of such a complex has not been demonstrated in mammalian cells. Coimmunoprecipitation of the endogenous hCAP-C-hCAP-E complex from HeLa extracts identified a 155-kDa protein interacting with hCAP-C-hCAP-E, termed condensation-related SMC-associated protein 1 (CNAP1). CNAP1 associates with mitotic chromosomes and is homologous to Xenopus condensin component XCAP-D2, indicating the presence of a condensin complex in human cells. Chromosome association of human condensin is mitosis specific, and the majority of condensin dissociates from chromosomes and is sequestered in the cytoplasm throughout interphase. However, a subpopulation of the complex was found to remain on chromosomes as foci in the interphase nucleus. During late G(2)/early prophase, the larger nuclear condensin foci colocalize with phosphorylated histone H3 clusters on partially condensed regions of chromosomes. These results suggest that mitosis-specific function of human condensin may be regulated by cell cycle-specific subcellular localization of the complex, and the nuclear condensin that associates with interphase chromosomes is involved in the reinitiation of mitotic chromosome condensation in conjunction with phosphorylation of histone H3.

• Hopfner KP et al.
• Structural biology of Rad50 ATPase: ATP-driven conformational control in DNA double-strand break repair and the ABC-ATPase superfamily.
• Cell. 2000; 101: 789-800
• Display abstract

• To clarify the key role of Rad50 in DNA double-strand break repair (DSBR), we biochemically and structurally characterized ATP-bound and ATP-free Rad50 catalytic domain (Rad50cd) from Pyrococcus furiosus. Rad50cd displays ATPase activity plus ATP-controlled dimerization and DNA binding activities. Rad50cd crystal structures identify probable protein and DNA interfaces and reveal an ABC-ATPase fold, linking Rad50 molecular mechanisms to ABC transporters, including P glycoprotein and cystic fibrosis transmembrane conductance regulator. Binding of ATP gamma-phosphates to conserved signature motifs in two opposing Rad50cd molecules promotes dimerization that likely couples ATP hydrolysis to dimer dissociation and DNA release. These results, validated by mutations, suggest unified molecular mechanisms for ATP-driven cooperativity and allosteric control of ABC-ATPases in DSBR, membrane transport, and chromosome condensation by SMC proteins.

• Parisi S et al.
• Rec8p, a meiotic recombination and sister chromatid cohesion phosphoprotein of the Rad21p family conserved from fission yeast to humans.
• Mol Cell Biol. 1999; 19: 3515-28
• Display abstract

• Our work and that of others defined mitosis-specific (Rad21 subfamily) and meiosis-specific (Rec8 subfamily) proteins involved in sister chromatid cohesion in several eukaryotes, including humans. Mutation of the fission yeast Schizosaccharomyces pombe rec8 gene was previously shown to confer a number of meiotic phenotypes, including strong reduction of recombination frequencies in the central region of chromosome III, absence of linear element polymerization, reduced pairing of homologous chromosomes, reduced sister chromatid cohesion, aberrant chromosome segregation, defects in spore formation, and reduced spore viability. Here we extend the description of recombination reduction to the central regions of chromosomes I and II. We show at the protein level that expression of rec8 is meiosis specific and that Rec8p localizes to approximately 100 foci per prophase nucleus. Rec8p was present in an unphosphorylated form early in meiotic prophase but was phosphorylated prior to meiosis I, as demonstrated by analysis of the mei4 mutant blocked before meiosis I. Evidence for the persistence of Rec8p beyond meiosis I was obtained by analysis of the mutant mes1 blocked before meiosis II. A human gene, which we designate hrec8, showed significant primary sequence similarity to rec8 and was mapped to chromosome 14. High mRNA expression of mouse and human rec8 genes was found only in germ line cells, specifically in testes and, interestingly, in spermatids. hrec8 was also expressed at a low level in the thymus. Sequence similarity and testis-specific expression indicate evolutionarily conserved functions of Rec8p in meiosis. Possible roles of Rec8p in the integration of different meiotic events are discussed.

• Sutani T, Yuasa T, Tomonaga T, Dohmae N, Takio K, Yanagida M
• Fission yeast condensin complex: essential roles of non-SMC subunits for condensation and Cdc2 phosphorylation of Cut3/SMC4.
• Genes Dev. 1999; 13: 2271-83
• Display abstract

• The condensin complex in frog extracts, containing two SMC (structural maintenance of chromosomes) and three non-SMC subunits, promotes mitotic chromosome condensation, and its supercoiling activity increases during mitosis by Cdc2 phosphorylation. Here, we report that fission yeast has the same five-member condensin complex, each of which is essential for mitotic condensation. The condensin complex was purified and the subunits were identified by microsequencing. Cnd1, Cnd2, and Cnd3, three non-SMC subunits showing a high degree of sequence conservation to frog subunits, are essential for viability, and their gene disruption leads to a phenotype indistinguishable from that observed in cut3-477 and cut14-208, known mutations in SMC4 and SMC2-like subunits. Condensin subunits tagged with GFP were observed to alter dramatically their localization during the cell cycle, enriched in the nucleus during mitosis, but cytoplasmic during other stages. This stage-specific alteration in localization requires mitosis-specific phosphorylation of the T19 Cdc2 site in Cut3. The T19 site is phosphorylated in vitro by Cdc2 kinase and shows the maximal phosphorylation in metaphase in vivo. Its alanine substitution mutant fails to suppress the temperature-sensitive phenotype of cut3-477, and shows deficiency in condensation, probably because Cut3 T19A remains cytoplasmic. Therefore, direct Cdc2 phosphorylation of fission yeast condensin may facilitate its nuclear accumulation during mitosis.

• Paques F, Haber JE
• Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae.
• Microbiol Mol Biol Rev. 1999; 63: 349-404
• Display abstract

• The budding yeast Saccharomyces cerevisiae has been the principal organism used in experiments to examine genetic recombination in eukaryotes. Studies over the past decade have shown that meiotic recombination and probably most mitotic recombination arise from the repair of double-strand breaks (DSBs). There are multiple pathways by which such DSBs can be repaired, including several homologous recombination pathways and still other nonhomologous mechanisms. Our understanding has also been greatly enriched by the characterization of many proteins involved in recombination and by insights that link aspects of DNA repair to chromosome replication. New molecular models of DSB-induced gene conversion are presented. This review encompasses these different aspects of DSB-induced recombination in Saccharomyces and attempts to relate genetic, molecular biological, and biochemical studies of the processes of DNA repair and recombination.

• Bhatt AM et al.
• The DIF1 gene of Arabidopsis is required for meiotic chromosome segregation and belongs to the REC8/RAD21 cohesin gene family.
• Plant J. 1999; 19: 463-72
• Display abstract

• Cohesins are a group of conserved proteins responsible for cohesion between replicated sister chromatids during mitosis and meiosis and which are implicated in double-strand break repair and meiotic recombination. We describe here the identification and characterisation of an Arabidopsis gene - DETERMINATE, INFERTILE1 (DIF1), which is a homolog of the Schizosaccharomyces pombe REC8/RAD21 cohesin genes, and is essential for meiotic chromosome segregation. Five independent alleles of the DIF1 gene were isolated by transposon mutagenesis, and the mutants show complete male and female sterility. Pollen mother cells (PMCs) of dif1 mutants show multiple meiotic defects which are represented by univalent chromosomes and chromosome fragmentation at metaphase I, and acentric fragments and chromatin bridges in meiosis I and II. Consequently, chromosome segregation is strongly affected, resulting in meiotic products of uneven size, shape and of variable ploidy. The similarities in phenotype, and the sequence homology between DIF1 and the REC8/RAD21 cohesins suggests that cohesin function is largely conserved between eukaryotes and highlights the essential role cohesins play in plant meiosis.

• Wilson S, Tavassoli M, Watts FZ
• Schizosaccharomyces pombe rad32 protein: a phosphoprotein with an essential phosphoesterase motif required for repair of DNA double strand breaks.
• Nucleic Acids Res. 1998; 26: 5261-9
• Display abstract

• The Schizosaccharomyces pombe Rad32 protein is required for repair of DNA double strand breaks, minichromosome stability and meiotic recombination. We show here that the Rad32 protein is phosphorylated in a cell cycle-dependent manner and during meiosis. The phosphorylation is not dependent on the checkpoint protein Rad3. Analysis of a partially purified protein preparation indicates that Rad32 is likely to act in a complex. Characterisation of the rad32-1 mutation and site-directed mutagenesis indicate that three aspartate residues in the conserved phosphoesterase motifs are important for both mitotic and meiotic functions, namely response to UV and ionising radiation and spore viability.

• Wu L et al.
• Miz1, a novel zinc finger transcription factor that interacts with Msx2 and enhances its affinity for DNA.
• Mech Dev. 1997; 65: 3-17
• Display abstract

• Msx2 is a homeobox gene with a regulatory role in inductive tissue interactions, including those that pattern the skull. We demonstrated previously that individuals affected with an autosomal dominant disorder of skull morphogenesis (craniosynostosis, Boston type) bear a mutated form of Msx2 in which a histidine is substituted for a highly conserved proline in position 7 of the N-terminal arm of the homeodomain (p148h). The mutation behaves as a dominant positive in transgenic mice. The location of the mutation in the N-terminal arm of the homeodomain, a region which in other homeodomain proteins plays a key part in protein-protein interactions, prompted us to undertake a yeast two hybrid screen for Msx2-interacting proteins. Here we present a functional analysis of one such protein, designated Miz1 (Msx-interacting-zinc finger). Miz1 is a zinc finger-containing protein whose amino acid sequence closely resembles that of the yeast protein, Nfi-1. Together these proteins define a new, highly conserved protein family. Analysis of Miz1 expression by Northern blot and in situ hybridization revealed a spatiotemporal pattern that overlaps that of Msx2. Further, Miz1 is a sequence specific DNA binding protein, and it can function as a positive-acting transcription factor. Miz1 interacts directly with Msx2 in vitro and enhances the DNA binding affinity of Msx2 for a functionally important element in the rat osteocalcin promoter. The p148h mutation in Msx2 augments the Miz1 effect on Msx2 DNA binding, suggesting a reason why this mutation behaves in vivo as a dominant positive, and providing a potential explanation of the craniosynostosis phenotype.

• Willson J, Wilson S, Warr N, Watts FZ
• Isolation and characterization of the Schizosaccharomyces pombe rhp9 gene: a gene required for the DNA damage checkpoint but not the replication checkpoint.
• Nucleic Acids Res. 1997; 25: 2138-46
• Display abstract

• Checkpoint controls exist in eukaryotic cells to ensure that cells do not enter mitosis in the presence of DNA damage or unreplicated chromosomes. In Schizosaccharomyces pombe many of the checkpoint genes analysed to date are required for both the DNA damage and the replication checkpoints, an exception being chk1 . We report here on the characterization of nine new methylmethane sulphonate (MMS)-sensitive S.pombe mutants, one of which is defective in the DNA damage checkpoint but not the replication checkpoint. We have cloned and sequenced the corresponding gene. The predicted protein is most similar to the Saccharomyces cerevisiae Rad9 protein, having 46% similarity and 26% identity. The S.pombe protein, which we have named Rhp9 (Rad9 homologue in S. pombe) on the basis of structural and phenotypic similarity, also contains motifs present in BRCA1 and 53BP1. Deletion of the gene is not lethal and results in a DNA damage checkpoint defect. Epistasis analysis with other S.pombe checkpoint mutants indicates that rhp9 acts in a process involving the checkpoint rad genes and that the rhp9 mutant is phenotypically very similar to chk1.

• Okayama H, Nagata A, Jinno S, Murakami H, Tanaka K, Nakashima N
• Cell cycle control in fission yeast and mammals: identification of new regulatory mechanisms.
• Adv Cancer Res. 1996; 69: 17-62

• Gilbreth M et al.
• The highly conserved skb1 gene encodes a protein that interacts with Shk1, a fission yeast Ste20/PAK homolog.
• Proc Natl Acad Sci U S A. 1996; 93: 13802-7
• Display abstract

• The Shk1 protein kinase, a homolog of Saccharomyces cerevisiae Ste20 and mammalian p21Cdc42/Rac-activated kinases, is an essential component of a Ras- and Cdc42-dependent signaling cascade required for cell viability, normal morphology, and mitogen-activated protein kinase-mediated sexual responses in the fission yeast, Schizosaccharomyces pombe. To identify S. pombe proteins that modulate or mediate Shk1 functions, we conducted a two-hybrid screen for Shk1-interacting proteins. One of the genes identified as a result of this screen was skb1. We show that Skb1 interacts with a region of the N-terminal regulatory domain of Shk1 distinct from that to which Cdc42 binds, and that Shk1, Cdc42, and Skb1 are able to form a ternary complex in vivo. S.pombe cells carrying an skb1 null mutation are less elongate in morphology than wild-type cells and exhibit a moderate growth defect. The morphology defect of the skb1 deletion mutant is suppressed by overexpression of Shk1. Overexpression of Skb1 causes wild-type S. pombe cells to become hyperelongated. Additional genetic analyses described herein suggest that Skb1 is a component of the morphology control branch of the Ras signaling cascade in S. pombe and that it positively modulates Shk1 function. Homologs of Skb1 are encoded by open reading frames in the genomes of S. cerevisiae and Caenorhabditis elegans and by an uncharacterized human cDNA sequence. Thus, skb1 may be the first well-characterized member of a highly conserved family of genes encoding potential p21Cdc42/Rac-activated kinase regulators.

• McCollum D, Balasubramanian MK, Pelcher LE, Hemmingsen SM, Gould KL
• Schizosaccharomyces pombe cdc4+ gene encodes a novel EF-hand protein essential for cytokinesis.
• J Cell Biol. 1995; 130: 651-60
• Display abstract

• Schizosaccharomyces pombe cells divide by medial fission. One class of cell division mutants (cdc), the late septation mutants, defines four genes: cdc3, cdc4, cdc8, and cdc12 (Nurse, P., P. Thuriaux, and K. Nasmyth. 1976. Mol. & Gen. Genet. 146:167-178). We have cloned and characterized the cdc4 gene and show that the predicted gene product. Cdc4p, is a 141-amino acid polypeptide that is similar in sequence to EF-hand proteins including myosin light chains, calmodulin, and troponin C. Two temperature-sensitive lethal alleles, cdc4-8 and cdc4-31, accumulate multiple nuclei and multiple improper F-actin rings and septa but fail to complete cytokinesis. Deletion of cdc4 also results in a lethal terminal phenotype characterized by multinucleate, elongated cells that fail to complete cytokinesis. Sequence comparisons suggest that Cdc4p may be a member of a new class of EF-hand proteins. Cdc4p localizes to a ringlike structure in the medial region of cells undergoing cytokinesis. Thus, Cdc4p appears to be an essential component of the F-actin contractile ring. We find that Cdc4 protein forms a complex with a 200-kD protein which can be cross-linked to UTP, a property common to myosin heavy chains. Together these results suggest that Cdc4p may be a novel myosin light chain.

• Hirano T, Mitchison TJ
• A heterodimeric coiled-coil protein required for mitotic chromosome condensation in vitro.
• Cell. 1994; 79: 449-58
• Display abstract

• We report here a chromosomal protein that plays an essential role in mitotic chromosome condensation in Xenopus egg extracts. Two polypeptides, designated XCAP-C and XCAP-E, were found to associate with each other in the extracts, presumably forming a heterodimer. During chromosome assembly in mitotic extracts, XCAP-C/E was recruited to the chromatin and formed a discrete internal structure within assembled chromosomes. Antibody blocking experiments showed that XCAP-C function is required for both assembly and structural maintenance of mitotic chromosomes in vitro. Deduced amino acid sequences revealed that the two polypeptides share common structural motifs, consisting of an N-terminal NTP-binding domain, two central coiled-coil regions, and a C-terminal conserved domain. These motifs are highly conserved in a protein family, members of which have been identified recently in both prokaryotes and eukaryotes.

• Saitoh N, Goldberg IG, Wood ER, Earnshaw WC
• ScII: an abundant chromosome scaffold protein is a member of a family of putative ATPases with an unusual predicted tertiary structure.
• J Cell Biol. 1994; 127: 303-18
• Display abstract

• Here, we describe the cloning and characterization of ScII, the second most abundant protein after topoisomerase II, of the chromosome scaffold fraction to be identified. ScII is structurally related to a protein, Smc1p, previously found to be required for accurate chromosome segregation in Saccharomyces cerevisiae. ScII and the other members of the emerging family of SMC1-like proteins are likely to be novel ATPases, with NTP-binding A and B sites separated by two lengthy regions predicted to form an alpha-helical coiled-coil. Analysis of the ScII B site predicted that ScII might use ATP by a mechanism similar to the bacterial recN DNA repair and recombination enzyme. ScII is a mitosis-specific scaffold protein that colocalizes with topoisomerase II in mitotic chromosomes. However, ScII appears not to be associated with the interphase nuclear matrix. ScII might thus play a role in mitotic processes such as chromosome condensation or sister chromatid disjunction, both of which have been previously shown to involve topoisomerase II.

• Harper JW, Adami GR, Wei N, Keyomarsi K, Elledge SJ
• The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases.
• Cell. 1993; 75: 805-16
• Display abstract

• The cyclin-dependent kinase Cdk2 associates with cyclins A, D, and E and has been implicated in the control of the G1 to S phase transition in mammals. To identify potential Cdk2 regulators, we have employed an improved two-hybrid system to isolate human genes encoding Cdk-interacting proteins (Cips). CIP1 encodes a novel 21 kd protein that is found in cyclin A, cyclin D1, cyclin E, and Cdk2 immunoprecipitates. p21CIP1 is a potent, tight-binding inhibitor of Cdks and can inhibit the phosphorylation of Rb by cyclin A-Cdk2, cyclin E-Cdk2, cyclin D1-Cdk4, and cyclin D2-Cdk4 complexes. Cotransfection experiments indicate that CIP1 and SV40 T antigen function in a mutually antagonistic manner to control cell cycle progression.

• Tabata S, Hotta Y, Stern H
• General recombination in meiosis.
• Tokai J Exp Clin Med. 1986; 11: 395-401
• Display abstract

• The meiotic events of homologous pairing and chiasma formation are apparently related to a number of meiotic processes. These include a delayed replication of small portion of genome (zygoDNA). L-protein, present transitionally in meiotic S-phase and leptotene, can bind to the specific sequences of zygo DNAs to form a nick and selectively suppress their replication until zygotene. Other meiosis specific proteins (R-protein, U-protein, rec A-like protein (m-rec) probably related with pairing and recombination but none of these, either alone or combination, is capable of inducing recombination in vitro. We have isolated proteins from meiotic and somatic cells that are capable of recombining two DNA plasmids in vitro. They have been designated m-recombinase and s-recombinase, respectively. m-recombinase appears during meiotic prophase with or without chiasma-formation, suggesting that regulation of meiotic recombination depends, not on the formation of appropriate enzymes but on rendering the enzymes effective by altering chromatin-structure. s-recombinase activity seems to be related to cell division and/or cell differentiation. The assay system used here for necessary recombination may be applicable to various situations.

• Tas S
• Cellular aging, neoplastic transformation, meiotic rejuvenation, and the structure of chromatin complex.
• Monogr Dev Biol. 1984; 17: 178-92

• Lindsley DL, Sandler L
• The genetic analysis of meiosis in female Drosophila melanogaster.
• Philos Trans R Soc Lond B Biol Sci. 1977; 277: 295-312
• Display abstract

• The three major features of meiosis are first synapsis, then exchange, and finally, disjunction of homologous chromosomes; these phenomena occur before pachytene, during pachytene, and after pachytene respectively. The effects of meiotic mutants, or other perturbations, either endogenous or exogenous, on the meiotic process may be assigned tentatively to one of these intervals, based on the earliest discernible abnormality. Thus mutants exhibiting abnormal disjunction and normal exchange affect post-pachytene functions; mutants exhibiting abnormal disjunction and exchange but with ultrastructurally normal appearing synaptonemal complex affect pachytene functions; and mutants with abnormal disjunction, exchange, and synaptonemal complex affect prepachytene functions. This rationale is applied to the temporal seriation of effects of meiotic mutants and chromosomal abnormalities on the meiotic programme.

• Humphreys GO, Willshaw GA, Smith HR, Anderson ES
• Mutagenesis of plasmid DNA with hydroxylamine: isolation of mutants of multi-copy plasmids.
• Mol Gen Genet. 1976; 145: 101-8
• Display abstract

• An investigation of in vitro mutagenesis of plasmid DNA with hydroxylamine is described. The treated plasmid DNA was used to transform Escherichia coli K12. Mutants of the plasmid NTP3, which codes for resistance to ampicillin and sulphonamides, were isolated and characterised. They were classified according to the reduction in level of their beta-lactamase activity. Hydroxylamine-induced mutants of NTP14 were also isolated. This plasmid codes for ampicillin resistance, synthesis of colicin E1, and the EcoRI restriction and modification enzymes. One class of mutants is lethal to the host strain at temperatures above 33 degrees C, but carrier strains grow well at 28 degrees C. There is evidence that these mutants code for a temperature-sensitive EcoRI modification activity: the lethal effect probably results from the cleavage of the host-cell DNA by the restriction enzyme at non-permissive temperatures. The possible genetic uses of the mutant plasmids for the production of hybrid plasmids in the bacterial cell are discussed.

• Hotchkiss RD
• Molecular basis for genetic recombination.
• Genetics. 1974; 78: 247-57

• Fabre F
• The role of repair mechanisms in the variations of ultraviolet and gamma-radiation sensitivity during the cell cycle of Schizosaccharomyces pombe.
• Radiat Res. 1973; 56: 528-39

• Threlkeld SF, Stoltz JM
• A genetic analysis of non-disjunction and mitotic recombination in Neurospora crassa.
• Genet Res. 1970; 16: 29-35

• Davies DR
• The control of dark repair mechanisms in meiotic cells.
• Mol Gen Genet. 1967; 100: 140-9

• HURST DD, FOGEL S
• MITOTIC RECOMBINATION AND HETEROALLELIC REPAIR IN SACCHAROMYCES CEREVISIAE.
• Genetics. 1964; 50: 435-58

• MAGNE F
• [Meiosis and number of chromosomes in the Laminariacea (Laminariales, Pheophyceae)].
• C R Hebd Seances Acad Sci. 1953; 236: 515-7

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