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NORMAL GENOMIC

• Gilbert N, Allan J
• Supercoiling in DNA and chromatin.
• Curr Opin Genet Dev. 2014; 25: 15-21
• Display abstract

• Supercoiling is a fundamental property of DNA and chromatin. It is modulated by polymerase and topoisomerase activities and, through regulated constraint, by DNA/chromatin binding proteins. As a non-covalent and elusive topological modification, supercoiling has proved intractable to research despite being a crucial regulator of nuclear structure and function. Recent studies have improved our understanding of the formation, regulation and organisation of supercoiling domains in vivo, and reinforce the prospect that the propagation of supercoiling can influence local and global chromatin structure. However, to further our understanding the development of new experimental tools and models are required to better dissect the mechanics of this key topological regulator.

• Feldmann EA, Galletto R
• The DNA-binding domain of yeast Rap1 interacts with double-stranded DNA in multiple binding modes.
• Biochemistry. 2014; 53: 7471-83
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• Saccharomyces cerevisiae repressor-activator protein 1 (Rap1) is an essential protein involved in multiple steps of DNA regulation, as an activator in transcription, as a repressor at silencer elements, and as a major component of the shelterin-like complex at telomeres. All the known functions of Rap1 require the known high-affinity and specific interaction of the DNA-binding domain with its recognition sequences. In this work, we focus on the interaction of the DNA-binding domain of Rap1 (Rap1(DBD)) with double-stranded DNA substrates. Unexpectedly, we found that while Rap1(DBD) forms a high-affinity 1:1 complex with its DNA recognition site, it can also form lower-affinity complexes with higher stoichiometries on DNA. These lower-affinity interactions are independent of the presence of the recognition sequence, and we propose they originate from the ability of Rap1(DBD) to bind to DNA in two different binding modes. In one high-affinity binding mode, Rap1(DBD) likely binds in the conformation observed in the available crystal structures. In the other alternative lower-affinity binding mode, we propose that a single Myb-like domain of the Rap1(DBD) makes interactions with DNA, allowing for more than one protein molecule to bind to the DNA substrates. Our findings suggest that the Rap1(DBD) does not simply target the protein to its recognition sequence but rather it might be a possible point of regulation.

• Driessen RP et al.
• Crenarchaeal chromatin proteins Cren7 and Sul7 compact DNA by inducing rigid bends.
• Nucleic Acids Res. 2013; 41: 196-205
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• Archaeal chromatin proteins share molecular and functional similarities with both bacterial and eukaryotic chromatin proteins. These proteins play an important role in functionally organizing the genomic DNA into a compact nucleoid. Cren7 and Sul7 are two crenarchaeal nucleoid-associated proteins, which are structurally homologous, but not conserved at the sequence level. Co-crystal structures have shown that these two proteins induce a sharp bend on binding to DNA. In this study, we have investigated the architectural properties of these proteins using atomic force microscopy, molecular dynamics simulations and magnetic tweezers. We demonstrate that Cren7 and Sul7 both compact DNA molecules to a similar extent. Using a theoretical model, we quantify the number of individual proteins bound to the DNA as a function of protein concentration and show that forces up to 3.5 pN do not affect this binding. Moreover, we investigate the flexibility of the bending angle induced by Cren7 and Sul7 and show that the protein-DNA complexes differ in flexibility from analogous bacterial and eukaryotic DNA-bending proteins.

• Chen C, Gorlatova N, Herzberg O
• Pliable DNA conformation of response elements bound to transcription factor p63.
• J Biol Chem. 2012; 287: 7477-86
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• We show that changes in the nucleotide sequence alter the DNA conformation in the crystal structures of p63 DNA-binding domain (p63DBD) bound to its response element. The conformation of a 22-bp canonical response element containing an AT spacer between the two half-sites is unaltered compared with that containing a TA spacer, exhibiting superhelical trajectory. In contrast, a GC spacers abolishes the DNA superhelical trajectory and exhibits less bent DNA, suggesting that increased GC content accompanies increased double helix rigidity. A 19-bp DNA, representing an AT-rich response element with overlapping half-sites, maintains superhelical trajectory and reveals two interacting p63DBD dimers crossing one another at 120 degrees . p63DBD binding assays to response elements of increasing length complement the structural studies. We propose that DNA deformation may affect promoter activity, that the ability of p63DBD to bind to superhelical DNA suggests that it is capable of binding to nucleosomes, and that overlapping response elements may provide a mechanism to distinguish between p63 and p53 promoters.

• Sharma A, Jenkins KR, Heroux A, Bowman GD
• Crystal structure of the chromodomain helicase DNA-binding protein 1 (Chd1) DNA-binding domain in complex with DNA.
• J Biol Chem. 2011; 286: 42099-104
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• Chromatin remodelers are ATP-dependent machines that dynamically alter the chromatin packaging of eukaryotic genomes by assembling, sliding, and displacing nucleosomes. The Chd1 chromatin remodeler possesses a C-terminal DNA-binding domain that is required for efficient nucleosome sliding and believed to be essential for sensing the length of DNA flanking the nucleosome core. The structure of the Chd1 DNA-binding domain was recently shown to consist of a SANT and SLIDE domain, analogous to the DNA-binding domain of the ISWI family, yet the details of how Chd1 recognized DNA were not known. Here we present the crystal structure of the Saccharomyces cerevisiae Chd1 DNA-binding domain in complex with a DNA duplex. The bound DNA duplex is straight, consistent with the preference exhibited by the Chd1 DNA-binding domain for extranucleosomal DNA. Comparison of this structure with the recently solved ISW1a DNA-binding domain bound to DNA reveals that DNA lays across each protein at a distinct angle, yet contacts similar surfaces on the SANT and SLIDE domains. In contrast to the minor groove binding seen for Isw1 and predicted for Chd1, the SLIDE domain of the Chd1 DNA-binding domain contacts the DNA major groove. The majority of direct contacts with the phosphate backbone occur only on one DNA strand, suggesting that Chd1 may not strongly discriminate between major and minor grooves.

• Bermudez I, Garcia-Martinez J, Perez-Ortin JE, Roca J
• A method for genome-wide analysis of DNA helical tension by means of psoralen-DNA photobinding.
• Nucleic Acids Res. 2010; 38: 182-182
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• The helical tension of chromosomal DNA is one of the epigenetic landmarks most difficult to examine experimentally. The occurrence of DNA crosslinks mediated by psoralen photobinding (PB) stands as the only suitable probe for assessing this problem. PB is affected by chromatin structure when is done to saturation; but it is mainly determined by DNA helical tension when it is done to very low hit conditions. Hence, we developed a method for genome-wide analysis of DNA helical tension based on PB. We adjusted in vitro PB conditions that discern DNA helical tension and applied them to Saccharomyces cerevisiae cells. We selected the in vivo cross-linked DNA sequences and identified them on DNA arrays. The entire procedure was robust. Comparison of PB obtained in vivo with that obtained in vitro with naked DNA revealed that numerous chromosomal regions had deviated PB values. Similar analyses in yeast topoisomerase mutants uncovered further PB alterations across specific chromosomal domains. These results suggest that distinct chromosome compartments might confine different levels of DNA helical tension in yeast. Genome-wide analysis of psoralen-DNA PB can be, therefore, a useful approach to uncover a trait of the chromosome architecture not amenable to other techniques.

• McGuire AT, Keates RA, Cook S, Mangroo D
• Structural modeling identified the tRNA-binding domain of Utp8p, an essential nucleolar component of the nuclear tRNA export machinery of Saccharomyces cerevisiae.
• Biochem Cell Biol. 2009; 87: 431-43
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• Utp8p is an essential 80 kDa intranuclear tRNA chaperone that transports tRNAs from the nucleolus to the nuclear tRNA export receptors in Saccharomyces cerevisiae. To help understand the mechanism of Utp8p function, predictive tools were used to derive a partial model of the tertiary structure of Utp8p. Secondary structure prediction, supported by circular dichroism measurements, indicated that Utp8p is divided into 2 domains: the N-terminal beta sheet and the C-terminal alpha helical domain. Tertiary structure prediction was more challenging, because the amino acid sequence of Utp8p is not directly homologous to any known protein structure. The tertiary structures predicted by threading and fold recognition had generally modest scores, but for the C-terminal domain, threading and fold recognition consistently pointed to an alpha-alpha superhelix. Because of the sequence diversity of this fold type, no single structural template was an ideal fit to the Utp8p sequence. Instead, a composite template was constructed from 3 different alpha-alpha superhelix structures that gave the best matches to different portions of the C-terminal domain sequence. In the resulting model, the most conserved sequences grouped in a tight cluster of positive charges on a protein that is otherwise predominantly negative, suggesting that the positive-charge cleft may be the tRNA-binding site. Mutations of conserved positive residues in the proposed binding site resulted in a reduction in the affinity of Utp8p for tRNA both in vivo and in vitro. Models were also derived for the 10 fungal homologues of Utp8p, and the localization of the positive charges on the conserved surface was found in all cases. Taken together, these data suggest that the positive-charge cleft of the C-terminal domain of Utp8p is involved in tRNA-binding.

• Stefanovsky VY, Moss T
• The cruciform DNA mobility shift assay: a tool to study proteins that recognize bent DNA.
• Methods Mol Biol. 2009; 543: 537-46
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• So-called architectural DNA binding proteins such as those of the HMGB-box family induce DNA bending and kinking. However, these proteins often display only a weak sequence preference, making the analysis of their DNA binding characteristics difficult if not impossible in a standard electrophoretic mobility assay (EMSA). In contrast, such proteins often bind prebent DNAs with high affinity and specificity. A synthetic cruciform DNA structure will often provide an ideal binding site for such proteins, allowing their affinities for both bent and linear DNAs to be directly and simply determined by a modified form of EMSA.

• Ucar D, Beyer A, Parthasarathy S, Workman CT
• Predicting functionality of protein-DNA interactions by integrating diverse evidence.
• Bioinformatics. 2009; 25: 13744-13744
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• Chromatin immunoprecipitation (ChIP-chip) experiments enable capturing physical interactions between regulatory proteins and DNA in vivo. However, measurement of chromatin binding alone is not sufficient to detect regulatory interactions. A detected binding event may not be biologically relevant, or a known regulatory interaction might not be observed under the growth conditions tested so far. To correctly identify physical interactions between transcription factors (TFs) and genes and to determine their regulatory implications under various experimental conditions, we integrated ChIP-chip data with motif binding sites, nucleosome occupancy and mRNA expression datasets within a probabilistic framework. This framework was specifically tailored for the identification of functional and non-functional DNA binding events. Using this, we estimate that only 50% of condition-specific protein-DNA binding in budding yeast is functional. We further investigated the molecular factors determining the functionality of protein-DNA interactions under diverse growth conditions. Our analysis suggests that the functionality of binding is highly condition-specific and highly dependent on the presence of specific cofactors. Hence, the joint analysis of both, functional and non-functional DNA binding, may lend important new insights into transcriptional regulation.

• Dang W, Kagalwala MN, Bartholomew B
• The Dpb4 subunit of ISW2 is anchored to extranucleosomal DNA.
• J Biol Chem. 2007; 282: 19418-25
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• Histone fold proteins Dpb4 and Dls1 are components of the yeast ISW2 chromatin remodeling complex that resemble the smaller subunits of the CHRAC (Chromatin Accessibility Complex) complex found in Drosophila and humans. DNA photoaffinity labeling found that the Dpb4 subunit contacts extranucleosomal DNA 37-53 bp away from the entry/exit site of the nucleosome. Binding of Dpb4 to Isw2 and Itc2, the two largest subunits of ISW2, was found to require Dls1. Even after remodeling and nucleosome movement, Dpb4 tends to remain bound to its original binding site and likely serves as an anchor point for ISW2 on DNA. In vitro, only minor differences can be detected in the nucleosome binding and mobilization properties of ISW2 with or without Dpb4 and Dls1. Changes in the contacts of the largest subunit Itc1 with extranucleosomal DNA have, however, been found upon deletion of the Dpb4 and Dls1 dimer that may affect the nucleosome spacing properties of ISW2.

• Bellizzi JJ 3rd, Sorger PK, Harrison SC
• Crystal structure of the yeast inner kinetochore subunit Cep3p.
• Structure. 2007; 15: 1422-30
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• In budding yeast, the four-protein CBF3 complex (Skp1p-Ctf13p-Cep3p-Ndc10p) initiates kinetochore assembly by binding to the CDEIII locus of centromeric DNA. A Cep3p dimer recruits a Skp1p-Ctf13p heterodimer and contacts two sites on CDEIII. We report here the crystal structure, determined at 2.8 A resolution by multiple isomorphous replacement with anomalous scattering, of a truncated Cep3p (Cep3p [47-608]), comprising all but an N-terminal, Zn(2)Cys(6)-cluster, DNA-binding module. Cep3p has a well-ordered structure throughout essentially all of its polypeptide chain, unlike most yeast transcription factors, including those with Zn(2)Cys(6) clusters, such as Gal4p. This difference may reflect an underlying functional distinction: whereas any particular transcription factor must adapt to a variety of upstream activating sites, Cep3p scaffolds kinetochore assembly on centromeres uniformly configured on all 16 yeast chromosomes. We have, using the structure of Cep3p (47-608) and the known structures of Zn(2)Cys(6)-cluster domains, modeled the interaction of Cep3p with CDEIII.

• Bauerle KT, Kamau E, Grove A
• Interactions between N- and C-terminal domains of the Saccharomyces cerevisiae high-mobility group protein HMO1 are required for DNA bending.
• Biochemistry. 2006; 45: 3635-45
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• The Saccharomyces cerevisiae high-mobility group protein HMO1 is composed of two DNA-binding domains termed box A and box B, of which only box B is predicted to adopt a HMG fold, and a lysine-rich C-terminal extension. To assess the interaction between individual domains and their contribution to DNA binding, several HMO1 variants were analyzed. Using circular dichroism spectroscopy, thermal stability was measured. While the melting temperatures of HMO1-boxA and HMO1-boxB are 57.2 and 47.2 degrees C, respectively, HMO1-boxBC, containing box B and the entire C-terminal tail, melts at 46.1 degrees C, suggesting little interaction between box B and the tail. In contrast, full-length HMO1 exhibits a single melting transition at 47.9 degrees C, indicating that interaction between box A and either box B or the tail destabilizes this domain. As HMO1-boxAB, lacking only the lysine-rich C-terminal segment, exhibits two melting transitions at 46.0 and 63.3 degrees C, we conclude that the destabilization of the box A domain seen in full-length HMO1 is due primarily to its interaction with the lysine-rich tail. Determination of DNA substrate specificity using electrophoretic mobility shift assays shows unexpectedly that the lysine-rich tail does not increase DNA binding affinity but instead is required for DNA bending by full-length HMO1; HMO1-boxBC, lacking the box A domain, also fails to bend DNA. In contrast, both HMO1 and HMO1-boxAB, but not the individual HMG domains, exhibit preferred binding to constrained DNA minicircles. Taken together, our data suggest that interactions between box A and the C-terminal tail induce a conformation that is required for DNA bending.

• de Oliveira FM et al.
• Cloning the genes and DNA binding properties of High Mobility Group B1 (HMGB1) proteins from the human blood flukes Schistosoma mansoni and Schistosoma japonicum.
• Gene. 2006; 377: 33-45
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• The parasitic helminth Schistosoma mansoni contains three HMGB proteins, HMGB1, HMGB2 and HMGB3, of primary amino acid sequences highly similar to vertebrate proteins. In this report we describe the characterization of the HMGB1 proteins and their genes from S. mansoni and Schistosoma japonicum. The deduced amino acid sequences of HMGB1 proteins from both schistosome species are identical, and comprise 176 residues. The proteins contain the two evolutionarily highly conserved HMG-box domains, A and B, exhibiting 60% similarity to mammalian HMGB1. Unlike the human HMGB1 which contains an unbroken run of 30 glutamic or aspartic residues, the SmHMGB1 or SjHMGB1 proteins possess unusually short acidic C-terminal tails (5 acidic residues interrupted by 2 serines). Southern hybridization and DNA sequencing revealed a single copy HMGB1 gene, composed of 3 exons and two introns, in S. mansoni. The exon/intron boundaries are identical to those of the human HMGB1 gene, with the exception that the second exon of the SmHMGB1 gene which is not split into two exons as in the human HMGB1 gene. RNA blot analysis revealed that the SmHMGB1 gene is constitutively expressed in similar levels both in male and female worms. The single-sized mRNA for SmHMGB1 is consistent with the size derived from the cDNA. Although DNA binding properties of SmHMGB1 (or SjHMGB1) protein seem to be similar to those previously reported with human HMGB1, i.e., preferential binding to supercoiled DNA over linear DNA, specific recognition of DNA four-way junctions, DNA-induced supercoiling in the presence of topoisomerase I, and DNA bending, we have observed two important differences relative to those observed with the human HMGB1: (i) the inability of the isolated SmHMGB1 domain A to bend DNA (as revealed by T4 ligase-mediated circularization assay), and (ii) higher DNA supercoiling and bending potential of the SmHMGB1 protein as compared to its human counterpart. The latter finding may indicate that the long acidic C-tail of human HMGB1 has much stronger repressive role on DNA bending or DNA supercoiling by topoisomerase I at physiological ionic strength than the short C-tail of the SmHMGB1 protein. Considering the important role of HMGB1 in DNA replication, transcription, recombination, and in particularly, the mediation of inflammation responses in mammalian cells, further studies on schistosome HMGB proteins may provide valuable information related to schistosomiasis, where inflammation plays a critical role in this disease.

• Nourani A, Robert F, Winston F
• Evidence that Spt2/Sin1, an HMG-like factor, plays roles in transcription elongation, chromatin structure, and genome stability in Saccharomyces cerevisiae.
• Mol Cell Biol. 2006; 26: 1496-509
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• Spt2/Sin1 is a DNA binding protein with HMG-like domains that has been suggested to play a role in chromatin-mediated transcription in Saccharomyces cerevisiae. Previous studies have suggested models in which Spt2 plays an inhibitory role in the initiation of transcription of certain genes. In this work, we have taken several approaches to study Spt2 in greater detail. Our results have identified previously unknown genetic interactions between spt2Delta and mutations in genes encoding transcription elongation factors, including members of the PAF and HIR/HPC complexes. In addition, genome-wide and gene-specific chromatin immunoprecipitation analyses suggest that Spt2 is primarily associated with coding regions in a transcription-dependent fashion. Furthermore, our results show that Spt2, like other elongation factors, is required for the repression of transcription from a cryptic promoter within a coding region and that Spt2 is also required for repression of recombination within transcribed regions. Finally, we provide evidence that Spt2 plays a role in regulating the levels of histone H3 over transcribed regions. Taken together, our results suggest a direct link for Spt2 with transcription elongation, chromatin dynamics, and genome stability.

• Krajewski WA, Nakamura T, Mazo A, Canaani E
• A motif within SET-domain proteins binds single-stranded nucleic acids and transcribed and supercoiled DNAs and can interfere with assembly of nucleosomes.
• Mol Cell Biol. 2005; 25: 1891-9
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• The evolutionary conserved SET domain is present in many eukaryotic chromatin-associated proteins, including some members of the trithorax (TrxG) group and the polycomb (PcG) group of epigenetic transcriptional regulators and modifiers of position effect variegation. All SET domains examined exhibited histone lysine methyltransferase activity, implicating these proteins in the generation of epigenetic marks. However, the mode of the initial recruitment of SET proteins to target genes and the way that their association with the genes is maintained after replication are not known. We found that SET-containing proteins of the SET1 and SET2 families contain motifs in the pre-SET region or at the pre-SET-SET and SET-post-SET boundaries which very tightly bind single-stranded DNA (ssDNA) and RNA. These motifs also bind stretches of ssDNA generated by superhelical tension or during the in vitro transcription of duplex DNA. Importantly, such binding withstands nucleosome assembly, interfering with the formation of regular nucleosomal arrays. Two representatives of the SUV39 SET family, SU(VAR)3-9 and G9a, did not bind ssDNA. The trxZ11 homeotic point mutation, which is located within TRX SET and disrupts embryonic development, impairs the ssDNA binding capacity of the protein. We suggest that the motifs described here may be directly involved in the biological function(s) of SET-containing proteins. The binding of single-stranded nucleic acids might play a role in the initial recruitment of the proteins to target genes, in the maintenance of their association after DNA replication, or in sustaining DNA stretches in a single-stranded configuration to allow for continuous transcription.

• Park CJ, Lee JH, Choi BS
• Solution structure of the DNA-binding domain of RPA from Saccharomyces cerevisiae and its interaction with single-stranded DNA and SV40 T antigen.
• Nucleic Acids Res. 2005; 33: 4172-81
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• Replication protein A (RPA) is a three-subunit complex with multiple roles in DNA metabolism. DNA-binding domain A in the large subunit of human RPA (hRPA70A) binds to single-stranded DNA (ssDNA) and is responsible for the species-specific RPA-T antigen (T-ag) interaction required for Simian virus 40 replication. Although Saccharomyces cerevisiae RPA70A (scRPA70A) shares high sequence homology with hRPA70A, the two are not functionally equivalent. To elucidate the similarities and differences between these two homologous proteins, we determined the solution structure of scRPA70A, which closely resembled the structure of hRPA70A. The structure of ssDNA-bound scRPA70A, as simulated by residual dipolar coupling-based homology modeling, suggested that the positioning of the ssDNA is the same for scRPA70A and hRPA70A, although the conformational changes that occur in the two proteins upon ssDNA binding are not identical. NMR titrations of hRPA70A with T-ag showed that the T-ag binding surface is separate from the ssDNA-binding region and is more neutral than the corresponding part of scRPA70A. These differences might account for the species-specific nature of the hRPA70A-T-ag interaction. Our results provide insight into how these two homologous RPA proteins can exhibit functional differences, but still both retain their ability to bind ssDNA.

• Fazzio TG, Gelbart ME, Tsukiyama T
• Two distinct mechanisms of chromatin interaction by the Isw2 chromatin remodeling complex in vivo.
• Mol Cell Biol. 2005; 25: 9165-74
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• We have previously shown that Saccharomyces cerevisiae Isw2 complex slides nucleosomes to remodel chromatin in vivo. Our data suggested a model in which Isw2 complex binds the histone octamer and DNA separately to generate the force necessary for nucleosome movement. Here we find that the histone H4 "basic patch" is the only portion of any amino-terminal histone tail required for both target-specific association of Isw2 complex with chromatin and chromatin remodeling in vivo, whereas it is dispensable for basal levels of chromatin binding. Similarly, we find that nonremodeled chromatin structure and integrity of Isw2 complex are required only for target-specific association of Isw2 with chromatin. These data demonstrate fundamental differences between the target-specific and basal modes of chromatin binding by Isw2 complex in vivo and suggest that only the former involves contributions from DNA, histone H4, and sequence-specific DNA binding proteins. We propose a model for target recognition and chromatin remodeling by Isw2 complex in vivo.

• Morpurgo M, Radu A, Bayer EA, Wilchek M
• DNA condensation by high-affinity interaction with avidin.
• J Mol Recognit. 2004; 17: 558-66
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• Avidin, the basic biotin-binding glycoprotein from chicken egg white, is known to interact with DNA, whereas streptavidin, its neutral non-glycosylated bacterial analog, does not. In the present study we investigated the DNA-binding properties of avidin. Its affinity for DNA in the presence and absence of biotin was compared with that of other positively charged molecules, namely the protein lysozyme, the cationic polymers polylysine and polyarginine and an avidin derivative with higher isoelectric point (pI approximately 11) in which most of the lysine residues were converted to homoarginines. Gel-shift assays, transmission electron microscopy and dynamic light scattering experiments demonstrated an unexpectedly strong interaction between avidin and DNA. The most pronounced gel-shift retardation occurred with the avidin-biotin complex, followed by avidin alone and then guanidylated avidin. Furthermore, ultrastructural and light-scattering studies showed that avidin assembles on the DNA molecule in an organized manner. The assembly leads to the formation of nanoparticles that are about 50-100 nm in size (DNA approximately 5 kb) and have a rod-like or toroidal shape. In these particles the DNA is highly condensed and one avidin is bound to each 18 +/- 4 DNA base pairs. The complexes are very stable even at high dilution ([DNA] =10 pM) and are not disrupted in the presence of buffers or salt (up to 200 mM NaCl). The other positively charged molecules also condense DNA and form particles with a globular shape. However, in this case, these particles disassemble by dilution or in the presence of low salt concentration. The results indicate that the interaction of avidin with DNA may also occur under physiological conditions, further enhanced by the presence of biotin. This DNA-binding property of avidin may thus shed light on a potentially new physiological role for the protein in its natural environment.

• Adkins NL, Watts M, Georgel PT
• To the 30-nm chromatin fiber and beyond.
• Biochim Biophys Acta. 2004; 1677: 12-23
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• Chromatin fibers are intrinsically dynamic macromolecular complexes whose biological functions are intimately linked with their structure and interactions with chromatin-associated proteins (CAPs). Three-dimensional architectural transitions between or within the two co-existing chromatin types referred to as euchromatin and heterochromatin have been associated with activation or repression of nuclear functions. The presence of specific subsets of chromosomal proteins co-existing with the different chromatin conformations suggests a functional significance for their co-localization. The major points of emphasis of this review will assess the structure, function and recently documented exchanges amongst various members of the CAP family.

• Ali T, Coles P, Stevens TJ, Stott K, Thomas JO
• Two homologous domains of similar structure but different stability in the yeast linker histone, Hho1p.
• J Mol Biol. 2004; 338: 139-48
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• The Saccharomyces cerevisiae homologue of the linker histone H1, Hho1p, has two domains that are similar in sequence to the globular domain of H1 (and variants such as H5). It is an open question whether both domains are functional and whether they play similar structural roles. Preliminary structural studies showed that the two isolated domains, GI and GII, differ significantly in stability. In 10 mM sodium phosphate (pH 7), the GI domain, like the globular domains of H1 and H5, GH1 and GH5, was stably folded, whereas GII was largely unstructured. However, at high concentrations of large tetrahedral anions (phosphate, sulphate, perchlorate), which might mimic the charge-screening effects of DNA phosphate groups, GII was folded. In view of the potential significance of these observations in relation to the role of Hho1p, we have now determined the structures of its GI and GII domains by NMR spectroscopy under conditions in which GII (like GI) is folded. The backbone r.m.s.d. over the ordered residues is 0.43 A for GI and 0.97 A for GII. Both structures show the "winged-helix" fold typical of GH1 and GH5 and are very similar to each other, with an r.m.s.d. over the structured regions of 1.3 A, although there are distinct differences. The potential for GII to adopt a structure similar to that of GI when Hho1p is bound to chromatin in vivo suggests that both globular domains might be functional. Whether Hho1p performs a structural role by bridging two nucleosomes remains to be determined.

• Ray-Gallet D, Almouzni G
• DNA synthesis-dependent and -independent chromatin assembly pathways in Xenopus egg extracts.
• Methods Enzymol. 2004; 375: 117-31

• Ghosh S, Grove A
• Histone-like protein HU from Deinococcus radiodurans binds preferentially to four-way DNA junctions.
• J Mol Biol. 2004; 337: 561-71
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• The histone-like protein HU from Escherichia coli is involved in DNA compaction and in processes such as DNA repair and recombination. Its participation in these events is reflected in its ability to bend DNA and in its preferred binding to DNA junctions and DNA with single-strand breaks. Deinococcus radiodurans is unique in its ability to reconstitute its genome from double strand breaks incurred after exposure to ionizing radiation. Using electrophoretic mobility shift assays (EMSA), we show that D.radiodurans HU (DrHU) binds preferentially only to DNA junctions, with half-maximal saturation of 18 nM. In distinct contrast to E.coli HU, DrHU does not exhibit a marked preference for DNA with nicks or gaps compared to perfect duplex DNA, nor is it able to mediate circularization of linear duplex DNA. These unexpected properties identify DrHU as the first member of the HU protein family not to serve an architectural role and point to its potential participation in DNA recombination events. Our data also point to a mechanism whereby differential target site selection by HU proteins is achieved and suggest that the substrate specificity of HU proteins should be expected to vary as a consequence of their individual capacity for inducing the required DNA bend.

• Alessandri M, Beretta GL, Ferretti E, Mancia A, Khobta A, Capranico G
• Enhanced CPT sensitivity of yeast cells and selective relaxation of Ga14 motif-containing DNA by novel Gal4-topoisomerase I fusion proteins.
• J Mol Biol. 2004; 337: 295-305
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• Human topoisomerase I-B (Top1) efficiently relaxes DNA supercoils during basic cellular processes, and can be transformed into a DNA-damaging agent by antitumour drugs, enzyme mutations and DNA lesions. Here, we describe Gal4-Top1 chimeric proteins (GalTop) with an N-terminal truncation of Top1, and mutations of the Gal4 Zn-cluster and/or Top1 domains that impair their respective DNA-binding activities. Expression levels of chimeras were similar in yeast cells, however, GalTop conferred an increased CPT sensitivity to RAD52- yeast cells as compared to a GalTop with mutations of the Gal4 domain, showing that a functional Gal4 domain can alter in vivo functions of Top1. In vitro enzyme activity was tested with a DNA relaxation assay using negatively supercoiled plasmids with 0 to 5 Gal4 consensus motifs. Only GalTop with a functional Gal4 domain could direct DNA relaxation activity of Top1 specifically to DNA molecules containing Gal4 motifs. By using a substrate competition assay, we could demonstrate that the Gal4-anchored Top1 remains functional and efficiently relax DNA substrates in cis. The enhanced CPT sensitivity of GalTop in yeast cells may then be due to alterations of the chromatin-binding activity of Top1. The GalTop chimeras may indeed mimic a normal mechanism by which Top1 is recruited to chromatin sites in living cells. Such hybrid Top1s may be helpful in further dissecting enzyme functions, and constitute a prototype of a site-specific DNA cutter endowed with high cell lethality.

• Tsubota T, Maki S, Kubota H, Sugino A, Maki H
• Double-stranded DNA binding properties of Saccharomyces cerevisiae DNA polymerase epsilon and of the Dpb3p-Dpb4p subassembly.
• Genes Cells. 2003; 8: 873-88
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• BACKGROUND: DNA polymerase epsilon (Pol epsilon) of Saccharomyces cerevisiae participates in many aspects of DNA replication, as well as in DNA repair. In order to clarify molecular mechanisms employed in the multiple tasks of Pol epsilon, we have been characterizing the interaction between Pol epsilon and DNA. RESULTS: Analysis of the four-subunit Pol epsilon complex by gel mobility shift assay revealed that the complex binds not only to single-stranded (ss) DNA but also equally well to double-stranded (ds) DNA. A truncated polypeptide consisting of the N-terminal domain of Pol2p catalytic subunit binds to ssDNA but not to dsDNA, indicating that the Pol2p C-terminal domain and/or the auxiliary subunits are involved in the dsDNA-binding. The dsDNA-binding by Pol epsilon does not require DNA ends or specific DNA sequences. Further analysis by competition experiments indicated that Pol epsilon contains at least two distinct DNA-binding sites, one of which binds exclusively to ssDNA and the other to dsDNA. The dsDNA-binding site, however, is suggested to also bind ssDNA. The DNA polymerase activity of Pol epsilon is inhibited by ssDNA but not by dsDNA. Furthermore, purification of the Pol epsilon auxiliary subunits Dpb3p and Dpb4p revealed that these proteins form a heterodimer and associate with dsDNA. CONCLUSIONS: Pol epsilon has multiple sites at which it interacts with DNA. One of these sites has a strong affinity for dsDNA, a feature that is not generally associated with DNA polymerases. Involvement of the Dpb3p-Dpb4p complex in the dsDNA-binding of Pol epsilon is inferred.

• Brewer LR et al.
• Packaging of single DNA molecules by the yeast mitochondrial protein Abf2p.
• Biophys J. 2003; 85: 2519-24
• Display abstract

• Mitochondrial and nuclear DNA are packaged by proteins in a very different manner. Although protein-DNA complexes called "nucleoids" have been identified as the genetic units of mitochondrial inheritance in yeast and man, little is known about their physical structure. The yeast mitochondrial protein Abf2p was shown to be sufficient to compact linear dsDNA, without the benefit of supercoiling, using optical and atomic force microscopy single molecule techniques. The packaging of DNA by Abf2p was observed to be very weak as evidenced by a fast Abf2p off-rate (k(off) = 0.014 +/- 0.001 s(-1)) and the extremely small forces (<0.6 pN) stabilizing the condensed protein-DNA complex. Atomic force microscopy images of individual complexes showed the 190-nm structures are loosely packaged relative to nuclear chromatin. This organization may leave mtDNA accessible for transcription and replication, while making it more vulnerable to damage.

• Hopfner KP, Tainer JA
• Rad50/SMC proteins and ABC transporters: unifying concepts from high-resolution structures.
• Curr Opin Struct Biol. 2003; 13: 249-55
• Display abstract

• ATP-binding cassette (ABC)-type ATPases are chemo-mechanical engines for diverse biological pathways. ABC ATPase domains act not only in ABC transporters but also in DNA mismatch, nucleotide excision and double-strand break repair enzymes, as well as in chromosome segregation. Atomic-resolution crystal structures suggest molecular mechanisms for ABC ATPases and reveal surprisingly significant mechanistic and architectural conservation. This emerging unified structural biochemistry provides general medical and biological insights into how ABC proteins function as chemo-mechanical devices. ATP binding by the signature and Q-loop motifs drives the conformations of substrate-specific domains to accomplish diverse functions in transmembrane transport and DNA repair.

• Fallah S, Rabbani A
• Interaction of a low mobility group protein, LMG160, with deoxyribonucleic acid.
• Int J Biol Macromol. 2003; 31: 217-21
• Display abstract

• A fraction of low mobility group (LMG) nonhistone protein designated LMG(160) was isolated from rat liver chromatin by preparative gel electrophoresis and its interaction with DNA was studied using thermal denaturation and DNA-cellulose affinity chromatography techniques. The results showed that LMG(160) with an isoelecteric point of 5-5.5 was bound to DNA and decreased its melting temperature. Increasing ionic strengths decreased this effect. DNA-cellulose affinity chromatography showed the affinity of LMG(160) to double stranded DNA was higher than that to single stranded DNA, since it required 0.6 M NaCl for elution. The results suggest that LMG(160) protein preferentially binds to double stranded DNA destabilizes it and the binding is electrostatic.

• Montano SP, Pierce M, Cote ML, Vershon AK, Georgiadis MM
• Crystallographic studies of a novel DNA-binding domain from the yeast transcriptional activator Ndt80.
• Acta Crystallogr D Biol Crystallogr. 2002; 58: 2127-30
• Display abstract

• The Ndt80 protein is a transcriptional activator that plays a key role in the progression of the meiotic divisions in the yeast Saccharomyces cerevisiae. Ndt80 is strongly induced during the middle stages of the sporulation pathway and binds specifically to a promoter element called the MSE to activate transcription of genes required for the meiotic divisions. Here, the preliminary structural and functional studies to characterize the DNA-binding activity of this protein are reported. Through deletion analysis and limited proteolysis studies of Ndt80, a novel 32 kDa DNA-binding domain that is sufficient for DNA-binding in vitro has been defined. Crystals of the DNA-binding domain of Ndt80 in two distinct lattices have been obtained, for which diffraction data extend to 2.3 A resolution.

• Nakamura Y, Shimizu M, Yoshida M
• Distorted DNA structures induced by HMGB2 possess a high affinity for HMGB2.
• J Biochem. 2002; 131: 153-60
• Display abstract

• HMGB2 (HMG2) protein binds with DNA duplex in a sequence-nonspecific manner, then bends and unwinds the DNA. In DNA cyclization analyses for the bending activity of HMGB2, two unidentified bands, denoted alpha and beta, were observed in addition to monomer circular DNA (1C) on the gel. Re-electrophoresis and proteinase K digestion revealed that alpha and beta are complexes of circularized probe DNA (seeming 1C) with HMGB2 (K(d) approximately 10(-10) M). The DNA components of alpha and beta (alpha- and beta-DNA) showed higher affinities to HMGB2 than did the linear probe DNA (K(d) approximately 10(-7) M). The DNAs have distorted structures containing partial single-stranded regions. Nicked circular molecules presumably due to severe DNA distortion by HMGB2 were observed in alpha- and beta-DNA, in addition to closed circular double-stranded molecules. The alpha and beta bands were not formed in the presence of sole DNA binding regions which are necessary for DNA bending, indicating that the acidic C-tail in the HMGB2 molecule is necessary for inducing the peculiar distorted structures of higher affinity to HMGB2. HMGB2 binds with linker DNA and/or the entry and exit of nucleosomes fixed at both ends likewise mini-circles similar to alpha-DNA and beta-DNA. Thus, the distorted structures present in alpha-DNA and beta-DNA should be important in considering the functional mechanisms in which HMGB2 participates.

• Masse JE, Wong B, Yen YM, Allain FH, Johnson RC, Feigon J
• The S. cerevisiae architectural HMGB protein NHP6A complexed with DNA: DNA and protein conformational changes upon binding.
• J Mol Biol. 2002; 323: 263-84
• Display abstract

• NHP6A is a non-sequence-specific DNA-binding protein from Saccharomyces cerevisiae which belongs to the HMGB protein family. Previously, we have solved the structure of NHP6A in the absence of DNA and modeled its interaction with DNA. Here, we present the refined solution structures of the NHP6A-DNA complex as well as the free 15bp DNA. Both the free and bound forms of the protein adopt the typical L-shaped HMGB domain fold. The DNA in the complex undergoes significant structural rearrangement from its free form while the protein shows smaller but significant conformational changes in the complex. Structural and mutational analysis as well as comparison of the complex with the free DNA provides insight into the factors that contribute to binding site selection and DNA deformations in the complex. Further insight into the amino acid determinants of DNA binding by HMGB domain proteins is given by a correlation study of NHP6A and 32 other HMGB domains belonging to both the DNA-sequence-specific and non-sequence-specific families of HMGB proteins. The resulting correlations can be rationalized by comparison of solved structures of HMGB proteins.

• Pavlov NA, Cherny DI, Nazimov IV, Slesarev AI, Subramaniam V
• Identification, cloning and characterization of a new DNA-binding protein from the hyperthermophilic methanogen Methanopyrus kandleri.
• Nucleic Acids Res. 2002; 30: 685-94
• Display abstract

• Three novel DNA-binding proteins with apparent molecular masses of 7, 10 and 30 kDa have been isolated from the hyperthermophilic methanogen Methanopyrus kandleri. The proteins were identified using a blot overlay assay that was modified to emulate the high ionic strength intracellular environment of M.kandleri proteins. A 7 kDa protein, named 7kMk, was cloned and expressed in Escherichia coli. As indicated by CD spectroscopy and computer-assisted structure prediction methods, 7kMk is a substantially alpha-helical protein possibly containing a short N-terminal beta-strand. According to analytical gel filtration chromatography and chemical crosslinking, 7kMk exists as a stable dimer, susceptible to further oligomerization. Electron microscopy showed that 7kMk bends DNA and also leads to the formation of loop-like structures of approximately 43.5 +/- 3.5 nm (136 +/- 11 bp for B-form DNA) circumference. A topoisomerase relaxation assay demonstrated that looped DNA is negatively supercoiled under physiologically relevant conditions (high salt and temperature). A BLAST search did not yield 7kMk homologs at the amino acid sequence level, but based on a multiple alignment with ribbon-helix-helix (RHH) transcriptional regulators, fold features and self-association properties of 7kMk we hypothesize that it could be related to RHH proteins.

• Krzeslak A, Lipinska A
• Glycoproteins present in the fraction of chromatin proteins loosely bound to DNA from hamster, chicken and frog liver cells.
• Gen Physiol Biophys. 2001; 20: 145-55
• Display abstract

• There are numerous glycoproteins recognized by Concanavalin A (ConA) and Galanthus nivalis agglutinin (GNA) in 0.35 mol/l NaCl soluble fraction of chromatin proteins loosely bound to DNA from hamster, chicken and frog liver cells. Results of our detailed comparative analysis show a marked similarity between liver chromatin glycoproteins from the examined animals. The presence of similar chromatin glycoproteins in different animal species may indicate that they play an important universal role in the liver cells.

• Roca J
• Varying levels of positive and negative supercoiling differently affect the efficiency with which topoisomerase II catenates and decatenates DNA.
• J Mol Biol. 2001; 305: 441-50
• Display abstract

• Type II DNA topoisomerases catalyze the transport of one DNA double helix through another. Here, by using a non-hydrolyzable analog of ATP, I examined the single-step DNA transport preferences of the yeast type II topoisomerase bound to positively and negatively supercoiled DNA rings. I found that negative supercoiling favors decatenation of DNA rings more than positive supercoiling. Conversely, positive supercoiling favors the catenation and knotting of DNA rings more than negative supercoiling. This vectorial effect of DNA supercoiling handedness supports a model in which type II topoisomerases can recognize three DNA segments, and highlights a novel influence of DNA supercoiling in global DNA topology.

• Torshin IY
• Clustering amino acid contents of protein domains: biochemical functions of proteins and implications for origin of biological macromolecules.
• Front Biosci. 2001; 6: 112-112
• Display abstract

• Structural classes of protein domains correlate with their amino acid compositions. Several successful algorithms (that use only amino acid composition) have been elaborated for the prediction of structural class or potential biochemical significance. This work deals with dynamic classification (clustering) of the domains on the basis of their amino acid composition. Amino acid contents of domains from a non-redundant PDB set were clustered in 20-dimensional space of amino acid contents. Despite the variations of an empirical parameter and non-redundancy of the set, only one large cluster (tens-hundreds of proteins) surrounded by hundreds of small clusters (1-5 proteins), was identified. The core of the largest cluster contains at least 64% DNA (nucleotide)-interacting protein domains from various sources. About 90% of the proteins of the core are intracellular proteins. 83% of the DNA/nucleotide interacting domains in the core belong to the mixed alpha-beta folds (a+b, a/b), 14% are all-alpha (mostly helices) and all-beta (mostly beta-strands) proteins. At the same time, when core domains that belong to one organism (E.coli) are considered, over 80% of them prove to be DNA/nucleotide interacting proteins. The core is compact: amino acid contents of domains from the core lie in relatively narrow and specific ranges. The core also contains several Fe-S cluster-binding domains, amino acid contents of the core overlap with ferredoxin and CO-dehydrogenase clusters, the oldest known proteins. As Fe-S clusters are thought to be the first biocatalysts, the results are discussed in relation to contemporary experiments and models dealing with the origin of biological macromolecules. The origin of most primordial proteins is considered here to be a result of co-adsorption of nucleotides and amino acids on specific clays, followed by en-block polymerization of the adsorbed mixtures of amino acids.

• Ohyama T
• Intrinsic DNA bends: an organizer of local chromatin structure for transcription.
• Bioessays. 2001; 23: 708-15
• Display abstract

• DNA with a curved trajectory of its helix axis is called bent DNA, or curved DNA. Interestingly, biologically important DNA regions often contain this structure, irrespective of the origin of DNA. In the last decade, considerable progress has been made in clarifying one role of bent DNA in prokaryotic transcription and its mechanism of action. However, the role of bent DNA in eukaryotic transcription remains unclear. Our recent study raises the possibility that bent DNA is implicated in the "functional packaging" of transcriptional regulatory regions into chromatin. In this article, I review recent progress in bent DNA research in eukaryotic transcription, and summarize the history of bent DNA research and several subjects relevant to this theme. Finally, I propose a hypothesis that bent DNA structures that mimic a negative supercoil, or have a right-handed superhelical writhe, organize local chromatin infrastructure to help the very first interaction between cis-acting DNA elements and activators that trigger transcription.

• Keith KC, Fitzgerald-Hayes M
• CSE4 genetically interacts with the Saccharomyces cerevisiae centromere DNA elements CDE I and CDE II but not CDE III. Implications for the path of the centromere dna around a cse4p variant nucleosome.
• Genetics. 2000; 156: 973-81
• Display abstract

• Each Saccharomyces cerevisiae chromosome contains a single centromere composed of three conserved DNA elements, CDE I, II, and III. The histone H3 variant, Cse4p, is an essential component of the S. cerevisiae centromere and is thought to replace H3 in specialized nucleosomes at the yeast centromere. To investigate the genetic interactions between Cse4p and centromere DNA, we measured the chromosome loss rates exhibited by cse4 cen3 double-mutant cells that express mutant Cse4 proteins and carry chromosomes containing mutant centromere DNA (cen3). When compared to loss rates for cells carrying the same cen3 DNA mutants but expressing wild-type Cse4p, we found that mutations throughout the Cse4p histone-fold domain caused surprisingly large increases in the loss of chromosomes carrying CDE I or CDE II mutant centromeres, but had no effect on chromosomes with CDE III mutant centromeres. Our genetic evidence is consistent with direct interactions between Cse4p and the CDE I-CDE II region of the centromere DNA. On the basis of these and other results from genetic, biochemical, and structural studies, we propose a model that best describes the path of the centromere DNA around a specialized Cse4p-nucleosome.

• Janiak-Spens F, West AH
• Functional roles of conserved amino acid residues surrounding the phosphorylatable histidine of the yeast phosphorelay protein YPD1.
• Mol Microbiol. 2000; 37: 136-44
• Display abstract

• The histidine-containing phosphotransfer (HPt) protein YPD1 is an osmoregulatory protein in yeast that facilitates phosphoryl transfer between the two response regulator domains associated with SLN1 and SSK1. Based on the crystal structure of YPD1 and the sequence alignment of YPD1 with other HPt domains, we site-specifically engineered and purified several YPD1 mutants in order to examine the role of conserved residues surrounding the phosphorylatable histidine (H64). Substitution of the positively charged residues K67 and R90 destabilized the phospho-imidazole linkage, whereas substitution of G68 apparently reduces accessibility of H64. These findings, together with the effect of other mutations, provide biochemical support of the proposed functional roles of conserved amino acid residues of HPt domains.

• Schwanbeck R et al.
• Point mutations within AT-hook domains of the HMGI homologue HMGIYL1 affect binding to gene promoter but not to four-way junction DNA.
• Biochemistry. 2000; 39: 14419-25
• Display abstract

• High-mobility group I/Y (HMGI/Y) proteins are chromosomal proteins involved in gene and chromatin regulation. Elevated levels of HMGI/Y proteins were reported in diverse malignant tumors, and rearrangements of their genes are casually involved in the development of benign tumors. In humans, the chromosomal locus Xp22 has been often found to be affected in diverse benign mesenchymal tumors. Recent studies revealed that this region contains a retropseudogene HMGIYL1 which potentially can be activated in a way of "exonization" upon aberrations involving this region. The coding sequence of the HMGIY-L1 is highly homologous to the HMGI(Y) gene. On the protein level, both HMGIYL1 and HMGI differ at few amino acid residues, including their putative DNA-binding domains (DBDs). Here we have approached the question of whether the HMGIYL1 product would be able to adopt a role of HMGI in the context of binding to gene promoters and chromatin. Comparative binding studies, employing protein footprinting technique, revealed that HMGIYL1 has lost the ability to bind to the promoter of the interferon beta gene, but retained its high affinity for the four-way junction DNA. Our results stress the importance of particular residues within the DBDs for DNA binding and demonstrate that tight binding of HMGI/Y proteins to the four-way junction DNA can be achieved in alternative ways. The binding of HMGIYL1 to four-way junction DNA suggests that activation of the HMGIYL1 gene would yield a protein sharing some binding properties with HMG1-box proteins and histone H1. Thus, the HMGIYL1 could interplay together with these components in chromatin regulation.

• Li R
• Stimulation of DNA replication in Saccharomyces cerevisiae by a glutamine- and proline-rich transcriptional activation domain.
• J Biol Chem. 1999; 274: 30310-4
• Display abstract

• Glutamine-rich Sp1 and proline-rich CTF1, two extensively studied mammalian transcription factors, bind to origins of replication in DNA tumor viruses and stimulate viral DNA replication in mammalian cells. Here it is shown that, when tethered to a plasmid-borne cellular origin of replication, the activation domains of both proteins can enhance origin function in Saccharomyces cerevisiae. Hydrophobic patches in Sp1 and CTF1 that mediate transcriptional activation in higher eukaryotes are also important for activation of replication in yeast. However, only the activation domain of CTF1 can enhance initiation of replication from a chromosomally embedded origin. This correlates with the ability of CTF1 to alter the local chromatin structure around the chromosomal origin of replication. The CTF1-induced chromatin remodeling occurs at multiple stages of the cell cycle. These findings strongly suggest a high degree of conservation in the mechanisms used by various types of transcription factors to stimulate viral and cellular DNA replication in eukaryotes.

• Alche JD, Paul E, Dickinson H
• Heterologously expressed polypeptide from the yeast meiotic gene HOP1 binds preferentially to yeast DNA.
• Protein Expr Purif. 1999; 16: 251-60
• Display abstract

• HOP1 protein, present in sporulating cells of Saccharomyces cerevisiae and believed to be a component of the synaptonemal complex, has been expressed in Escherichia coli fused to a biotinylated tag protein. Once solubilized from bacterial inclusion bodies, the HOP1 fusion protein was purified by using a combination of avidin-affinity chromatography and gel filtration FPLC and refolded. Sequence comparisons indicate that the HOP1 gene product contains a zinc finger motif, which may confer DNA binding properties, and the recombinant polypeptide was used to assess the putative DNA binding properties of the product of native HOP1 protein using a gel-shift assay. Protein and protein-DNA complexes were detected by exploiting the affinity of streptavidin-alkaline phosphatase for the biotinylated tag protein after Western blotting. The HOP1 fusion protein bound unambiguously to digested genomic yeast DNA. This binding possessed some degree of specificity, was maintained under a wide range of salt concentrations, and was unaffected by the presence of high concentrations of competitor DNA (synthetic poly[dI-dC].poly[dI-dC]). In contrast, no shift was detected when the fusion protein was incubated with digested genomic DNA from Arabidopsis, or with lambda/HindIII DNA. Incubation with digested genomic DNA from Lilium produced a small change in the mobility of the protein. The biotinylated tag protein failed to show any DNA binding activity. Scatchard analysis indicated an apparent yeast genomic DNA:HOP1 fusion protein dissociation constant of K(d) = 5 x 10(-7) M.

• Livingstone-Zatchej M, Suter B, Thoma F
• Photolyase. A molecular tool to characterize chromatin structure in yeast.
• Methods Mol Biol. 1999; 119: 245-59

• Morse RH
• Analysis of DNA topology in yeast chromatin.
• Methods Mol Biol. 1999; 119: 379-93

• Akhmedov AT, Gross B, Jessberger R
• Mammalian SMC3 C-terminal and coiled-coil protein domains specifically bind palindromic DNA, do not block DNA ends, and prevent DNA bending.
• J Biol Chem. 1999; 274: 38216-24
• Display abstract

• The C-terminal domains of yeast structural maintenance of chromosomes (SMC) proteins were previously shown to bind double-stranded DNA, which generated the idea of the antiparallel SMC heterodimer, such as the SMC1/3 dimer, bridging two DNA molecules. Analysis of bovine SMC1 and SMC3 protein domains now reveals that not only the C-terminal domains, but also the coiled-coil region, binds DNA, while the N terminus is inactive. Duplex DNA and DNA molecules with secondary structures are highly preferred substrates for both the C-terminal and coiled-coil domains. Contrasting other cruciform DNA-binding proteins like HMG1, the SMC3 C-terminal and coiled-coil domains do not bend DNA, but rather prevent bending in ring closure assays. Phosphatase, exonuclease, and ligase assays showed that neither domain renders DNA ends inaccessible for other enzymes. These observations allow modifications of models for SMC-DNA interactions.

• King DA, Zhang L, Guarente L, Marmorstein R
• Structure of HAP1-18-DNA implicates direct allosteric effect of protein-DNA interactions on transcriptional activation.
• Nat Struct Biol. 1999; 6: 22-7
• Display abstract

• HAP1 is a yeast transcriptional activator that binds with equal affinity to the dissimilar upstream activation sequences UAS1 and UAS(CYC7), but activates transcription differentially when bound to each site. HAP1-18 harbors an amino acid change in the DNA binding domain. While binding UAS1 poorly, HAP1-18 binds UAS(CYC7) with wild-type properties and activates transcription at elevated levels relative to HAP1. We have determined the structure of HAP1-18-UAS(CYC7) and have compared it to HAP1-UAS(CYC7). Unexpectedly, the single amino acid substitution in HAP1-18 nucleates a significantly altered hydrogen bond interface between the protein and DNA resulting in DNA conformational changes and an ordering of one N-terminal arm of the protein dimer along the DNA minor groove. These observations, together with a large subset of transcriptionally defective mutations in the HAP1 DNA-binding domain that map to the HAP1-DNA interface, suggest that protein-DNA interactions may have direct allosteric effects on transcriptional activation.

• Bozhenok LN, Khazina EB, Chernolovskaya EL, Kobets ND
• Chromatin proteins surrounding the d(G-T)n repeats and polyamine influence as revealed by photoaffinity labeling with reactive pd(A-C)6 derivatives.
• FEBS Lett. 1998; 440: 38-40
• Display abstract

• The complementary-addressed modification of DNA and proteins in chromatin using photoreactive derivatives of pd(AC)6 has been studied. These oligonucleotides form complementary complexes with specific DNA sequences and modify both DNA and proteins in the vicinity of these regions, and can be used for investigation of the protein environment in DNA. We have demonstrated that photoreactive derivatives of oligonucleotides can quickly and efficiently modify chromatin proteins and seem to be promising for investigation of perturbations in chromatin structure during the cell cycle. A comparison between modified chromatin from synchronized cells has demonstrated differences in the sets of proteins modified in the S and G1/S phases of the cell cycle. An increase in spermine and spermidine concentrations leads to an increase in modification of definite chromatin proteins. It can be supposed that the B-Z transition that can be stabilized by the presence of natural polyamines is one of the reasons for the presence of single-stranded DNA regions, containing sets of (dG-dT)n and accessible for interaction with complementary oligonucleotides.

• Kjeldsen T et al.
• Secretory expression of human albumin domains in Saccharomyces cerevisiae and their binding of myristic acid and an acylated insulin analogue.
• Protein Expr Purif. 1998; 13: 163-9
• Display abstract

• Albumin is organized in three homologous domains formed by double loops stabilized by disulfide bonds. Utilizing a secretory expression system based on a synthetic secretory prepro-leader, the three human serum albumin domains were expressed in the yeast Saccharomyces cerevisiae. Human serum albumin domains I and III were efficiently expressed and secreted, indicating that these domains can form independent structural units capable of folding into stable tertiary structures. In contrast, albumin domain II was not secreted and disappeared early in the secretory pathway. Human serum albumin has the ability to bind a large number of small molecule ligands, including fatty acids, presumably due to its structure and structural flexibility. Purified albumin domain III bound myristic acid, whereas purified albumin domain I did not bind myristic acid. A new soluble long-acting insulin an alogue acylated with myristic acid (Markussen J., et al., Diabetologia 39, 281-288, 1996) bound to domain III and bound markedly more weakly to domain I.

• Levesque D, Veilleux S, Caron N, Boissonneault G
• Architectural DNA-binding properties of the spermatidal transition proteins 1 and 2.
• Biochem Biophys Res Commun. 1998; 252: 602-9
• Display abstract

• Mammalian spermiogenesis is characterized by replacement of somatic histones by a set of basic nuclear transition proteins thought to be actively involved in the chromatin remodeling process. The two major transition proteins of the elongating spermatids, namely TP1 and TP2, were expressed and purified using a bacterial expression system. Both topoisomerase and ligase-mediated supercoiling assays demonstrated that TP1, as well as TP2, did not produce detectable changes in the twist and/or writhe of DNA molecules upon binding. Ligase-mediated circularization assay further demonstrated that neither of the transition proteins under study produced bends in linear DNA but that they both have the capacity to stimulate oligomerization of linear DNA fragments. We further established that the transition proteins are in vitro substrates for the Ca+2-phospholipid-dependent protein kinase (PKC) as well as the cAMP-dependent protein kinase (PKA). PKC phosphorylation was found to strongly weaken the DNA-condensing ability of TP2. These results suggest that the major transition proteins represent architectural factors able to stabilize DNA in a nonsupercoiled state, thereby promoting DNA condensation.

• Zlatanova J, van Holde K
• Binding to four-way junction DNA: a common property of architectural proteins?
• FASEB J. 1998; 12: 421-31
• Display abstract

• Proteins that can be shown to strongly bind in vitro to the four-way (Holliday) junction DNA include not only the obvious candidates such as enzymes involved in recombination, but also a remarkably diverse group of seemingly unrelated proteins. These include the HMG1 box proteins, members of the HMGI-Y family, winged helix proteins (including linker histones), the SWI/SNF complex, and some totally unrelated prokaryotic proteins. What these proteins seem to share is a propensity to bind to bent DNA, to bend DNA upon binding, and/or to preferentially interact with DNA crossings. Thus, they appear to be, in the main, architectural proteins, although some (like the SWI/SNF complex) have very specific functional roles as well. Perhaps because they bind to or promote the formation of particular DNA structures, the four-way junction binding proteins are frequently interchangeable in cellular function. Furthermore, since a given kind of structure can be recognized by many different protein motifs, it is not surprising that apparently unrelated proteins can fall into such a single functional class.

• Tsen H, Levene SD
• Supercoiling-dependent flexibility of adenosine-tract-containing DNA detected by a topological method.
• Proc Natl Acad Sci U S A. 1997; 94: 2817-22
• Display abstract

• Intrinsically bent DNA sequences have been implicated in the activation of transcription by promoting juxtaposition of DNA sequences near the terminal loop of a superhelical domain. We have developed a novel topological assay for DNA looping based on lambda integrative recombination to study the effects of intrinsically bent DNA sequences on the tertiary structure of negatively supercoiled DNA. Remarkably, the localization of adenosine-tract (A-tract) sequences in the terminal loop of a supercoiled plasmid is independent of the extent of intrinsic bending. The results suggest that A-tract-containing sequences have other properties that organize the structure of superhelical domains apart from intrinsic bending and may explain the lack of conservation in the degree of A-tract-dependent bending among DNA sequences located upstream of bacterial promoters.

• Saxena P et al.
• Probing Flp: a new approach to analyze the structure of a DNA recognizing protein by combining the genetic algorithm, mutagenesis and non-canonical DNA target sites.
• Biochim Biophys Acta. 1997; 1340: 187-204
• Display abstract

• A topological and functional overview of a DNA recognition protein with unknown structure can be achieved by combining three different, but complementary approaches: modeling by the genetic algorithm, functional analysis of mutated variants, and testing the target DNA using non-canonical oligonucleotides. As an example we choose the Flp protein, a site-specific recombinase from Saccharomyces cerevisiae. We derive the topological outline including the DNA binding cleft, examine DNA binding regions by deletional and mutational analysis, and analyze the DNA binding site using 7-deazaadenine, 7-deazaguanine, inosine and 4-O-methylthymine as probes. The combined data offer a comprehensive sketch of a plausible protein architecture for Flp. The structure is detailed enough to verify the prediction accuracy for different peptide regions from pre-existing data and by new experimental design.

• Yona E, Bangio H, Erlich P, Tepper SH, Katcoff DJ
• The C-terminal domain of SIN1 in yeast interacts with a protein that binds the URS1 region of the yeast HO gene.
• Mol Gen Genet. 1995; 246: 774-7
• Display abstract

• A protein or protein complex has previously been identified in Saccharomyces cerevisiae which both binds a short DNA sequence in URS1 of HO and interacts with SIN1. SIN1, which has some sequence similarity to mammalian HMG1, is an abundant chromatin protein in yeast and is thought to participate in the transcriptional repression of a specific family of genes. SIN1 binds DNA weakly, though it has no DNA binding specificity. Here we address the nature of the interaction between SIN1 and the specific DNA binding protein(s) to HO DNA. We show that the isolated C-terminal region of SIN1 can interact in vitro with the DNA binding protein, causing a supershift in a gel mobility shift assay. Interestingly, inclusion of the region in SIN1 which contains two acidic sequences, precludes the binding of recombinant protein to the DNA/protein complex.

• Droge P
• Protein tracking-induced supercoiling of DNA: a tool to regulate DNA transactions in vivo?
• Bioessays. 1994; 16: 91-9
• Display abstract

• An interplay between DNA-dependent biological processes appears to be crucial for cell viability. At the molecular level, this interplay relies heavily on the communication between DNA-bound proteins, which can be facilitated and controlled by the dynamic structure of double-stranded DNA. Hence, DNA structural alterations are recognized as potential tools to transfer biological information over some distance within a genome. Until recently, however, direct evidence for DNA structural information as a mediator between cellular processes was lacking. This changed when the concept of transient waves of DNA supercoiling, induced by proteins tracking along the right-handed DNA double helix, came into the limelight. Indeed, a number of observations now suggest that helix tracking-induced DNA structural information might be exploited to participate in the regulation of a variety of DNA transactions in vivo.

• Kraevskii VA, Krylov DIu, Razin SV, Mikhailov VS
• [Analysis of the conformational mobility of DNA in transcriptionally-active chromatin].
• Biofizika. 1993; 38: 108-16
• Display abstract

• An attempt is made to estimate the degree of conformational flexibility of DNA in different fractions of nucleosomes of transcriptionally active chromatin of higher eukaryotes, and in yeast nucleosomes as well. We have used the circular dichroism method to estimate conformational changes in DNA induced by temperature shift. The DNA conformational potential was shown to be approximately the same in all the fractions of nucleosomes under study.

• Morse RH, Pederson DS, Dean A, Simpson RT
• Yeast nucleosomes allow thermal untwisting of DNA.
• Nucleic Acids Res. 1987; 15: 10311-30
• Display abstract

• Thermal untwisting of DNA is suppressed in vitro in nucleosomes formed with chicken or monkey histones. In contrast, results obtained for the 2 micron plasmid in Saccharomyces cerevisiae are consistent with only 30% of the DNA being constrained from thermal untwisting in vivo. In this paper, we examine thermal untwisting of several plasmids in yeast cells, nuclei, and nuclear extracts. All show the same quantitative degree of thermal untwisting, indicating that this phenomenon is independent of DNA sequence. Highly purified yeast plasmid chromatin also shows a large degree of thermal untwisting, whereas circular chromatin reconstituted using chicken histones is restrained from thermal untwisting in yeast nuclear extracts. Thus, the difference in thermal untwisting between yeast chromatin and that assembled with chicken histones is most likely due to differences in the constituent histone proteins.

• Soldatenkov VA, Filippovich IV, Romantsev EF
• [Nature of conformational restrictions in nuclear superhelical DNA from thymus lymphocytes].
• Biokhimiia. 1983; 48: 883-9
• Display abstract

• The lymphocyte nucleoids of mouse thymus contain about 40% of rapidly labelled nuclear RNA, about 9% of total intracellular protein and all nuclear DNA. Relaxation of superhelical DNA after thymocyte nucleoids treatment with pronase or RNAase suggests that non-histone proteins and/or RNAs are involved in conformational restrictions in the superhelical domains of cell DNAs. Thymocyte nucleoids proteins are represented by two groups of nonhistone proteins with molecular weights of 50 000-60 000 and 75 000-85 000. An essential role in the appearance of conformational restrictions in thymocyte superhelical DNA belongs to disulfide bonds.

• Lohr D, Hereford L
• Yeast chromatin is uniformly digested by DNase-I.
• Proc Natl Acad Sci U S A. 1979; 76: 4285-8
• Display abstract

• The DNase I (EC 3.1.21.1) sensitivity of transcribed yeast chromatin has been examined. We find that, in contrast to chromatin from higher eukaryotes, transcribed yeast chromatin and total yeast chromatin are equally sensitive to DNase I digestion. We interpret these results to mean that the entire yeast genome exists in a state that represents a restricted proportion of total chromatin in higher eukaryotes.

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