SMART MODE:

NORMAL GENOMIC

• Reeves R, Beckerbauer L
• HMGI/Y proteins: flexible regulators of transcription and chromatin structure.
• Biochim Biophys Acta. 2001; 1519: 13-29
• Display abstract

• The mammalian HMGI/Y (HMGA) non-histone proteins participate in a wide variety of cellular processes including regulation of inducible gene transcription, integration of retroviruses into chromosomes and the induction of neoplastic transformation and promotion of metastatic progression of cancer cells. Recent advances have contributed greatly to our understanding of how the HMGI/Y proteins participate in the molecular mechanisms underlying these biological events. All members of the HMGI/Y family of 'high mobility group' proteins are characterized by the presence of multiple copies of a conserved DNA-binding peptide motif called the 'AT hook' that preferentially binds to the narrow minor groove of stretches of AT-rich sequence. The mammalian HMGI/Y proteins have little, if any, secondary structure in solution but assume distinct conformations when bound to substrates such as DNA or other proteins. Their intrinsic flexibility allows the HMGI/Y proteins to participate in specific protein-DNA and protein-protein interactions that induce both structural changes in chromatin substrates and the formation of stereospecific complexes called 'enhanceosomes' on the promoter/enhancer regions of genes whose transcription they regulate. The formation of such regulatory complexes is characterized by reciprocal inductions of conformational changes in both the HMGI/Y proteins themselves and in their interacting substrates. It may well be that the inherent flexibility of the HMGI/Y proteins, combined with their ability to undergo reversible disordered-to-ordered structural transitions, has been a significant factor in the evolutionary selection of these proteins for their functional role(s) in cells.

• Krejci L, Damborsky J, Thomsen B, Duno M, Bendixen C
• Molecular dissection of interactions between Rad51 and members of the recombination-repair group.
• Mol Cell Biol. 2001; 21: 966-76
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• Recombination is important for the repair of DNA damage and for chromosome segregation during meiosis; it has also been shown to participate in the regulation of cell proliferation. In the yeast Saccharomyces cerevisiae, recombination requires products of the RAD52 epistasis group. The Rad51 protein associates with the Rad51, Rad52, Rad54, and Rad55 proteins to form a dynamic complex. We describe a new strategy to screen for mutations which cause specific disruption of the interaction between certain proteins in the complex, leaving other interactions intact. This approach defines distinct protein interaction domains and protein relationships within the Rad51 complex. Alignment of the mutations onto the constructed three-dimensional model of the Rad51 protein reveal possible partially overlapping interfaces for the Rad51-Rad52 and the Rad51-Rad54 interactions. Rad51-Rad55 and Rad51-Rad51 interactions are affected by the same spectrum of mutations, indicating similarity between the two modes of binding. Finally, the detection of a subset of mutations within Rad51 which disrupt the interaction with mutant Rad52 protein but activate the interaction with Rad54 suggests that dynamic changes within the Rad51 protein may contribute to an ordered reaction process.

• Encinar JA et al.
• Human p8 is a HMG-I/Y-like protein with DNA binding activity enhanced by phosphorylation.
• J Biol Chem. 2001; 276: 2742-51
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• We have studied the biochemical features, the conformational preferences in solution, and the DNA binding properties of human p8 (hp8), a nucleoprotein whose expression is affected during acute pancreatitis. Biochemical studies show that hp8 has properties of the high mobility group proteins, HMG-I/Y. Structural studies have been carried out by using circular dichroism (near- and far-ultraviolet), Fourier transform infrared, and NMR spectroscopies. All the biophysical probes indicate that hp8 is monomeric (up to 1 mm concentration) and partially unfolded in solution. The protein seems to bind DNA weakly, as shown by electrophoretic gel shift studies. On the other hand, hp8 is a substrate for protein kinase A (PKA). The phosphorylated hp8 (PKAhp8) has a higher content of secondary structure than the nonphosphorylated protein, as concluded by Fourier transform infrared studies. PKAhp8 binds DNA strongly, as shown by the changes in circular dichroism spectra, and gel shift analysis. Thus, although there is not a high sequence homology with HMG-I/Y proteins, hp8 can be considered as a HMG-I/Y-like protein.

• Schwanbeck R, Manfioletti G, Wisniewski JR
• Architecture of high mobility group protein I-C.DNA complex and its perturbation upon phosphorylation by Cdc2 kinase.
• J Biol Chem. 2000; 275: 1793-801
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• The high mobility group I-C (HMGI-C) protein is an abundant component of rapidly proliferating undifferentiated cells. High level expression of this protein is characteristic for early embryonic tissue and diverse tumors. HMGI-C can function as an architectural factor enhancing the activity of transcription factor NF-kappaB on the beta-interferon promoter. The protein has three minor groove DNA-binding domains (AT-hooks). Here, we describe the complex of HMGI-C with a fragment of the beta-interferon promoter. We show that the protein binds to NRDI and PRDII elements of the promoter with its first and second AT-hook, respectively. Phosphorylation by Cdc2 kinase leads to a partial derailing of the AT-hooks from the minor groove, affecting mainly the second binding domain. In contrast, binding to long AT stretches of DNA involves contacts with all three AT-hooks and is marginally sensitive to phosphorylation. Our data stress the importance of conformation of the DNA binding site and protein phosphorylation for its function.

• Rottgers K, Krohn NM, Lichota J, Stemmer C, Merkle T, Grasser KD
• DNA-interactions and nuclear localisation of the chromosomal HMG domain protein SSRP1 from maize.
• Plant J. 2000; 23: 395-405
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• The structure-specific recognition protein 1 (SSRP1) is a member of the protein family containing a high mobility group (HMG) domain DNA-binding motif. We have functionally characterised the 71.4 kDa Zm-SSRP1 protein from maize. The chromatin-associated Zm-SSRP1 is detected by immunoblot analysis in maize leaves, kernels and suspension culture cells, but not in roots. Mediated by its HMG domain, recombinant Zm-SSRP1 interacts structure-specifically with supercoiled DNA and DNA minicircles when compared with linear DNA. In linear duplex DNA, the protein does not recognise a specific sequence, but it binds preferentially to sequences containing the deformable dinucleotide TG, as demonstrated by a random oligonucleotide selection experiment. Zm-SSRP1 modulates DNA structure by bending the target sequence, since it promotes the circularisation of short DNA fragments in the presence of DNA ligase. Moreover, Zm-SSRP1 facilitates the formation of nucleoprotein structures, as measured using the bacterial site-specific beta-mediated recombination reaction. Analysis of the subcellular localisation of various SSRP1-GFP fusions revealed that, in contrast to HMG domain transcription factors, the nuclear localisation sequence of Zm-SSRP1 is situated within a 20-amino acid residue region adjacent to the HMG domain rather than within the DNA-binding domain. The results are discussed in the context of the likely function of SSRP1 proteins in transcription and replication.

• Taylor IA et al.
• Characterization of the DNA-binding domains from the yeast cell-cycle transcription factors Mbp1 and Swi4.
• Biochemistry. 2000; 39: 3943-54
• Display abstract

• The minimal DNA-binding domains of the Saccharomyces cerevisiae transcription factors Mbp1 and Swi4 have been identified and their DNA binding properties have been investigated by a combination of methods. An approximately 100 residue region of sequence homology at the N-termini of Mbp1 and Swi4 is necessary but not sufficient for full DNA binding activity. Unexpectedly, nonconserved residues C-terminal to the core domain are essential for DNA binding. Proteolysis of Mbp1 and Swi4 DNA-protein complexes has revealed the extent of these sequences, and C-terminally extended molecules with substantially enhanced DNA binding activity compared to the core domains alone have been produced. The extended Mbp1 and Swi4 proteins bind to their cognate sites with similar affinity [K(A) approximately (1-4) x 10(6) M(-)(1)] and with a 1:1 stoichiometry. However, alanine substitution of two lysine residues (116 and 122) within the C-terminal extension (tail) of Mbp1 considerably reduces the apparent affinity for an MCB (MluI cell-cycle box) containing oligonucleotide. Both Mbp1 and Swi4 are specific for their cognate sites with respect to nonspecific DNA but exhibit similar affinities for the SCB (Swi4/Swi6 cell-cycle box) and MCB consensus elements. Circular dichroism and (1)H NMR spectroscopy reveal that complex formation results in substantial perturbations of base stacking interactions upon DNA binding. These are localized to a central 5'-d(C-A/G-CG)-3' region common to both MCB and SCB sequences consistent with the observed pattern of specificity. Changes in the backbone amide proton and nitrogen chemical shifts upon DNA binding have enabled us to experimentally define a DNA-binding surface on the core N-terminal domain of Mbp1 that is associated with a putative winged helix-turn-helix motif. Furthermore, significant chemical shift differences occur within the C-terminal tail of Mbp1, supporting the notion of two structurally distinct DNA-binding regions within these proteins.

• 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
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• 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.

• Vautard G, Cotton P, Fevre M
• The glucose repressor CRE1 from Sclerotinia sclerotiorum is functionally related to CREA from Aspergillus nidulans but not to the Mig proteins from Saccharomyces cerevisiae.
• FEBS Lett. 1999; 453: 54-8
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• We isolated the putative glucose repressor gene cre1 from the phytopathogenic fungus Sclerotinia sclerotiorum. cre1 encodes a 429 amino acid protein 59% similar to the carbon catabolite repressor CREA from Aspergillus nidulans. In addition to the overall amino acid sequence relatedness between CRE1 and CREA proteins, cre1 can functionally complement the A. nidulans creAd30 mutation as assessed by repression of the alcohol dehydrogenase I gene expression. The CREI region carrying the two zinc fingers is also very similar to the DNA binding domains of the Saccharomyces cerevisiae glucose repressors Mig1p and Mig2p. Despite the presence in the CRE1 protein of several motifs involved in the regulation of Miglp activity, cre1 cannot complement mig deficiencies in S. cerevisiae. These data suggest that glucose repression pathways may have evolved differently in yeasts and filamentous fungi.

• Zhang XM, Verdine GL
• A small region in HMG I(Y) is critical for cooperation with NF-kappaB on DNA.
• J Biol Chem. 1999; 274: 20235-43
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• The high mobility group HMG I(Y) protein has been reported to promote the expression of several NF-kappaB-dependent genes by enhancing the binding of NF-kappaB to DNA. The molecular origins of cooperativity in the binding of NF-kappaB and HMG I(Y) to DNA are not well understood. Here we have examined the determinants of specificity in the binding of HMG I(Y), both alone and in cooperation with NF-kappaB, to two different DNA elements, PRDII from the interferon-beta enhancer and IgkappaB from the immunoglobulin kappa light chain enhancer. Of particular interest was the influence of a flanking AT-rich sequence on binding by HMG I(Y). Utilizing yeast one-hybrid screening assays together with alanine-scanning mutagenesis, we have identified mutations of residues in HMG I(Y) that decrease cooperative binding of NF-kappaB to PRDII and IgkappaB sites. These same mutations similarly decreased the binding of HMG I(Y) alone to DNA, and paradoxically, decreased the strength of protein-protein interactions between HMG I(Y) and NF-kappaB. Of the three tandemly repeated basic regions that represent putative DNA-binding motifs in HMG I(Y), the residues within the second repeat are most important for recognition of core NF-kappaB sites, whereas the second and third repeats both appear to be involved in binding to sites that are flanked by AT-rich sequences. Overall, the second repeat of HMG I(Y) is primarily responsible for the stimulatory effect of this protein on the binding of NF-kappaB to PRDII and IgkappaB elements.

• Saito K, Kikuchi T, Shirakawa H, Yoshida M
• The stabilized structural array of two HMG1/2-boxes endowed by a linker sequence between them is requisite for the effective binding of HMG1 with DNA.
• J Biochem (Tokyo). 1999; 125: 399-405
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• High mobility group (HMG) protein 1 contains two DNA binding motifs, called HMG1/2-boxes, linked with a linker region. The functional relationships between the two boxes and the mechanism of involvement of the linker region for effective binding of HMG1 were examined. The binding analyses of truncated HMG1 peptides with DNA indicated that the structural array of two boxes stabilizes the interaction of HMG1 with DNA. The mutation analyses of the linker region suggested that the region is equipped with tolerance for the deletion of a few amino acid residues to allow appropriate binding of the two boxes with DNA, and that the basic cluster in the linker sequence is in a position to interact with DNA. The existence of tolerance for the linker sequence was found to be conserved during the evolution of HMG1 protein homologues. A structural model for array of two boxes associating with DNA minor groove was constructed on the basis of the experimental results and energy minimization. The model proposes that the DNA binding region in HMG1 covers an 18 bp DNA region and induces its bending by about 140 degrees. The linker region may function to maintain the structural array of two HMG1/2-boxes by direct interaction with DNA.

• 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
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• 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.

• Vande Berg BJ, Sancar GB
• Evidence for dinucleotide flipping by DNA photolyase.
• J Biol Chem. 1998; 273: 20276-84
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• DNA photolyases repair pyrimidine dimers via a reaction in which light energy drives electron donation from a catalytic chromophore, FADH-, to the dimer. The crystal structure of Escherichia coli photolyase suggested that the pyrimidine dimer is flipped out of the DNA helix and into a cavity that leads from the surface of the enzyme to FADH-. We have tested this model using the Saccharomyces cerevisiae Phr1 photolyase which is >50% identical to E. coli photolyase over the region comprising the DNA binding domain. By using the bacterial photolyase as a starting point, we modeled the region encompassing amino acids 383-530 of the yeast enzyme. The model retained the cavity leading to FADH- as well as the band of positive electrostatic potential which defines the DNA binding surface. We found that alanine substitution mutations at sites within the cavity reduced both substrate binding and discrimination, providing direct support for the dinucleotide flip model. The roles of three residues predicted to interact with DNA flanking the dimer were also tested. Arg452 was found to be particularly critical to substrate binding, discrimination, and photolysis, suggesting a role in establishing or maintaining the dimer in the flipped state. A structural model for photolyase-dimer interaction is presented.

• Yen YM, Wong B, Johnson RC
• Determinants of DNA binding and bending by the Saccharomyces cerevisiae high mobility group protein NHP6A that are important for its biological activities. Role of the unique N terminus and putative intercalating methionine.
• J Biol Chem. 1998; 273: 4424-35
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• The non-histone proteins 6A/B (NHP6A/B) of Saccharomyces cerevisiae are high mobility group proteins that bind and severely bend DNA of mixed sequence. They exhibit high affinity for linear DNA and even higher affinity for microcircular DNA. The 16-amino acid basic segment located N-terminal to the high mobility group domain is required for stable complex formation on both linear and microcircular DNA. Although mutants lacking the N terminus are able to promote microcircle formation and Hin invertasome assembly at high protein concentrations, they are unable to form stable complexes with DNA, co-activate transcription, and complement the growth defect of Deltanhp6a/b mutants. A basic patch between amino acids 13 and 16 is critical for these activities, and a second basic patch between residues 8 and 10 is required for the formation of monomeric complexes with linear DNA. Mutational analysis suggests that proline 18 may direct the path of the N-terminal arm to facilitate DNA binding, whereas the conserved proline at position 21, tyrosine 28, and phenylalanine 31 function to maintain the tertiary structure of the high mobility group domain. Methionine 29, which may intercalate into DNA, is essential for NHP6A-induced microcircle formation of 75-bp but not 98-bp fragments in vitro, and for full growth complementation of Deltanhp6a/b mutants in vivo.

• Balaeff A, Churchill ME, Schulten K
• Structure prediction of a complex between the chromosomal protein HMG-D and DNA.
• Proteins. 1998; 30: 113-35
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• Non-histone chromosomal proteins are an important part of nuclear structure and function due to their ability to interact with DNA to form and modulate chromatin structure and regulate gene expression. However, the understanding of the function of chromosomal proteins at the molecular level has been hampered by the lack of structures of chromosomal protein-DNA complexes. We have carried out a molecular dynamics modeling study to provide insight into the mode of DNA binding to the chromosomal HMG-domain protein, HMG-D. Three models of a complex of HMG-D bound to DNA were derived through docking the protein to two different DNA fragments of known structure. Molecular dynamics simulations of the complexes provided data indicating the most favorable model. This model was further refined by molecular dynamics simulation and extensively analyzed. The structure of the corresponding HMG-D-DNA complex exhibits many features seen in the NMR structures of the sequence-specific HMG-domain-DNA complexes, lymphoid enhancer factor 1 (LEF-1) and testis determining factor (SRY). The model reveals differences from these known structures that suggest how chromosomal proteins bind to many different DNA sequences with comparable affinity.

• Frank O, Schwanbeck R, Wisniewski JR
• Protein footprinting reveals specific binding modes of a high mobility group protein I to DNAs of different conformation.
• J Biol Chem. 1998; 273: 20015-20
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• The high mobility group proteins I and Y (HMGI/Y) are abundant components of chromatin. They are thought to derepress chromatin, affect the assembly and activity of the transcriptional machinery, and associate with constitutive heterochromatin during mitosis. HMGI/Y protein molecules contain three potential DNA-binding motifs (AT-hooks), but the extent of contacts between DNA and the entire protein has not been determined. We have used a protein-footprinting procedure to map regions of the Chironomus HMGI protein molecule that are involved in contacts with DNA. We find that in the presence of double-stranded DNA all AT-hook motifs are protected against hydroxyl radical proteolysis. In contrast, only two motifs were protected in the presence of four-way junction DNA. Large regions that flank the AT-hook motifs were found to be strongly protected against proteolysis in complexes with interferon-beta promoter DNA, suggesting amino acid residues outside the AT-hooks considerably contribute to DNA binding.

• Huang DW, Fanti L, Pak DT, Botchan MR, Pimpinelli S, Kellum R
• Distinct cytoplasmic and nuclear fractions of Drosophila heterochromatin protein 1: their phosphorylation levels and associations with origin recognition complex proteins.
• J Cell Biol. 1998; 142: 307-18
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• The distinct structural properties of heterochromatin accommodate a diverse group of vital chromosome functions, yet we have only rudimentary molecular details of its structure. A powerful tool in the analyses of its structure in Drosophila has been a group of mutations that reverse the repressive effect of heterochromatin on the expression of a gene placed next to it ectopically. Several genes from this group are known to encode proteins enriched in heterochromatin. The best characterized of these is the heterochromatin-associated protein, HP1. HP1 has no known DNA-binding activity, hence its incorporation into heterochromatin is likely to be dependent upon other proteins. To examine HP1 interacting proteins, we isolated three distinct oligomeric species of HP1 from the cytoplasm of early Drosophila embryos and analyzed their compositions. The two larger oligomers share two properties with the fraction of HP1 that is most tightly associated with the chromatin of interphase nuclei: an underphosphorylated HP1 isoform profile and an association with subunits of the origin recognition complex (ORC). We also found that HP1 localization into heterochromatin is disrupted in mutants for the ORC2 subunit. These findings support a role for the ORC-containing oligomers in localizing HP1 into Drosophila heterochromatin that is strikingly similar to the role of ORC in recruiting the Sir1 protein to silencing nucleation sites in Saccharomyces cerevisiae.

• Webster CI, Packman LC, Pwee KH, Gray JC
• High mobility group proteins HMG-1 and HMG-I/Y bind to a positive regulatory region of the pea plastocyanin gene promoter.
• Plant J. 1997; 11: 703-15
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• A 268 bp region (P268) of the pea plastocyanin gene promoter responsible for high-level expression has been shown to interact with the high mobility group proteins HMG-1 and HMG-I/Y isolated from pea shoot chromatin. cDNAs encoding an HMG-1 protein of 154 amino acid residues containing a single HMG-box and a C-terminal acidic tail and an HMG-I/Y-like protein of 197 amino acid residues containing four AT-hooks have been isolated and expressed in Escherichia coli to provide large amounts of full-length proteins. DNase I footprinting identified eight binding sites for HMG-I/Y and six binding sites for HMG-1 in P268. Inhibition of binding by the antibiotic distamycin, which binds in the minor groove of A/T-rich DNA, revealed that HMG-I/Y binding was 400-fold more sensitive than HMG-1 binding. Binding-site selection from a pool of random oligonucleotides indicated that HMG-I/Y binds to oligonucleotides containing stretches of five or more A/T bp and HMG-1 binds preferentially to oligonucleotides enriched in dinucleotides such as TpT and TpG.

• Carlson DL, Skopp R, Price CM
• DNA-Binding properties of the replication telomere protein.
• Biochemistry. 1997; 36: 15900-8
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• The replication Telomere Protein, rTP, is a nuclear protein from the ciliate Euplotes crassus that appears to be a novel telomere replication factor. rTP shares extensive amino acid sequence identity with the two proteins that bind and protect the macronuclear telomeres from the ciliates Oxytricha and Euplotes. Since the most extended regions of conservation fall within the DNA-binding domains of the telomere-binding proteins, when rTP was first identified it was predicted to be another structural telomere-binding protein. However, subsequent research demonstrated that rTP transcripts accumulate only during DNA replication and the rTP protein localizes to the sites of DNA replication within Euplotes macronuclei. We have now expressed rTP in a heterologous expression system and have examined the DNA-binding properties of the recombinant protein. We show that rTP binds specifically to the G-strand of Euplotes telomeric DNA and hence has some of the same DNA-binding characteristics as the Euplotes and Oxytricha telomere-binding proteins. However, other aspects of rTP binding are unique. In particular, the protein exhibits a very high off-rate and can bind double-stranded DNA as well as internal tracts of telomeric sequence. We conclude that rTP and the telomere-binding proteins are members of a class of proteins that have a conserved DNA-binding motif tailored to bind the G-strand of telomeric DNA. However, the unique DNA-binding characteristics of rTP indicate that the protein has evolved to fulfil a specialized role during telomere replication.

• Yie J, Liang S, Merika M, Thanos D
• Intra- and intermolecular cooperative binding of high-mobility-group protein I(Y) to the beta-interferon promoter.
• Mol Cell Biol. 1997; 17: 3649-62
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• The mammalian high-mobility-group protein I(Y) [HMG I(Y)], while not a typical transcriptional activator, is required for the expression of many eukaryotic genes. HMG I(Y) appears to recruit and stabilize complexes of transcriptional activators through protein-DNA and protein-protein interactions. The protein binds to the minor groove of DNA via three short basic repeats, preferring tracts of adenines and thymines arranged on the same face of the DNA helix. However, the mode by which these three basic repeats function together to recognize HMG I(Y) binding sites has remained unclear. Here, using deletion mutants of HMG I(Y), DNase I footprinting, methylation interference, and in vivo transcriptional assays, we have characterized the binding of HMG I(Y) to the model beta-interferon enhancer. We show that two molecules of HMG I(Y) bind to the enhancer in a highly cooperative fashion, each molecule using a distinct pair of basic repeats to recognize the tandem AT-rich regions of the binding sites. We have also characterized the function of each basic repeat, showing that only the central repeat accounts for specific DNA binding and that the presence of a second repeat bound to an adjacent AT-rich region results in intramolecular cooperativity in binding. Surprisingly, the carboxyl-terminal acidic tail of HMG I(Y) is also important for specific binding in the context of the full-length protein. Our results present a detailed examination of HMG I(Y) binding in an important biological context, which can be extended not only to HMG I(Y) binding in other systems but also to the binding mode of many other proteins containing homologous basic repeats, which have been conserved from bacteria to humans.

• He S, Fox TD
• Membrane translocation of mitochondrially coded Cox2p: distinct requirements for export of N and C termini and dependence on the conserved protein Oxa1p.
• Mol Biol Cell. 1997; 8: 1449-60
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• To study in vivo the export of mitochondrially synthesized protein from the matrix to the intermembrane space, we have fused a synthetic mitochondrial gene, ARG8m, to the Saccharomyces cerevisiae COX2 gene in mitochondrial DNA. The Arg8mp moiety was translocated through the inner membrane when fused to the Cox2p C terminus by a mechanism dependent on topogenic information at least partially contained within the exported Cox2p C-terminal tail. The pre-Cox2p leader peptide did not signal translocation. Export of the Cox2p C-terminal tail, but not the N-terminal tail, was dependent on the inner membrane potential. The mitochondrial export system does not closely resemble the bacterial Sec translocase. However, normal translocation of both exported domains of Cox2p was defective in cells lacking the widely conserved inner membrane protein Oxa1p.

• Hill DA, Reeves R
• Competition between HMG-I(Y), HMG-1 and histone H1 on four-way junction DNA.
• Nucleic Acids Res. 1997; 25: 3523-31
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• High mobility group proteins HMG-I(Y) and HMG-1, as well as histone H1, all share the common property of binding to four-way junction DNA (4H), a synthetic substrate commonly used to study proteins involved in recognizing and resolving Holliday-type junctions formed during in vivo genetic recombination events. The structure of 4H has also been hypothesized to mimic the DNA crossovers occurring at, or near, the entrance and exit sites on the nucleosome. Furthermore, upon binding to either duplex DNA or chromatin, all three of these nuclear proteins share the ability to significantly alter the structure of bound substrates. In order to further elucidate their substrate binding abilities, electrophoretic mobility shift assays were employed to investigate the relative binding capabilities of HMG-I(Y), HMG-1 and H1 to 4H in vitro. Data indicate a definite hierarchy of binding preference by these proteins for 4H, with HMG-I(Y) having the highest affinity (Kd approximately 6.5 nM) when compared with either H1 (Kd approximately 16 nM) or HMG-1 (Kd approximately 80 nM). Competition/titration assays demonstrated that all three proteins bind most tightly to the same site on 4H. Hydroxyl radical footprinting identified the strongest site for binding of HMG-I(Y), and presumably for the other proteins as well, to be at the center of 4H. Together these in vitro results demonstrate that HMG-I(Y) and H1 are co-dominant over HMG-1 for binding to the central crossover region of 4H and suggest that in vivo both of these proteins may exert a dominant effect over HMG-1 in recognizing and binding to altered DNA structures, such as Holliday junctions, that have conformations similar to 4H.

• Maher JF, Nathans D
• Multivalent DNA-binding properties of the HMG-1 proteins.
• Proc Natl Acad Sci U S A. 1996; 93: 6716-20
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• HMG-I proteins are DNA-binding proteins thought to affect the formation and function of transcription complexes. Each protein contains three DNA-binding motifs, known as AT-hooks, that bind in the minor groove of AT tracts in DNA. Multiple AT-hooks within a polypeptide chain should contact multiple AT tracts, but the rules governing these interactions have not been defined. In this study, we demonstrate that high-affinity binding uses two or three appropriately spaced AT tracts as a single multivalent binding site. These principles have implications for binding to regulatory elements such as the interferon beta enhancer, TATA boxes, and serum response elements.

• Reeves R, Wolffe AP
• Substrate structure influences binding of the non-histone protein HMG-I(Y) to free nucleosomal DNA.
• Biochemistry. 1996; 35: 5063-74
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• High mobility group protein HMG-I(Y) selectively binds to stretches of A.T-rich B-form DNA in vitro by recognition of substrate structure rather nucleotide sequence. Recognition of altered DNA structures has also been proposed to explain the preferential binding of this non-histone protein to four-way junction DNA as well as to restricted regions of DNA on random-sequence nucleosome core particles. Here we describe experiments that examine the influence of intrinsic DNA structure, and of structure imposed by folding of DNA around histone cores, on the binding of HMG-I(Y). As substrates for binding, we chose defined-sequence DNA molecules containing A.T-rich segments demonstrated previously to have very different structures in solution. These segments are either intrinsically bent (phase A.T tracts), flexible (oligo[d(A-T)]), or straight and rigid [oligo(dA).oligo(dT)]. DNase-I and hydroxyl radical footprinting techniques were employed to analyze protein binding to these DNAs either free in solution or when they were reconstituted into monomer or dinucleosomes in vitro. Results indicate that the DNA structure exerts a significant influence on HMG-I(Y) binding both when substrates are free in solution and when they are wrapped into nucleosomal structures. For example, when DNA is free in solution, HMG-I(Y) prefers to bind to the narrow minor groove of A.T sequences but sometimes also binds to certain GpC residues having narrowed major grooves that are embedded in such sequences. On the other hand, depending on the structure and/or orientation assumed by particular A.T-rich segments on the surface of reconstituted histone octamers, HMG-I(Y) binding site selection on individual nucleosomes differs considerably. Two observations are of particular importance: (i) HMG-I(Y) can preferentially bind to certain types of A.T-DNA located on the surface of nucleosomes; and (ii) HMG-I(Y) binding can induce localized alterations in the helical periodicity and/or rotational setting of DNA on the surface of some nucleosomes. The abilities of HMG-I(Y) suggests that in vivo the protein may play an important role in recognizing and altering the structure of localized regions of chromatin.

• Xiao D, Huang K
• [Effect of phosphorylation by protein kinase C on the DNA binding activity of high mobility group protein I]
• Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 1996; 18: 79-83
• Display abstract

• High mobility group protein I (HMG-I) is a nonhistone chromosomal protein. The present study aims to examine phosphorylation of HMG-I by protein kinase C (PKC) and its effect on HMG-I's DNA binding activity. HMG-I, extracted and purified from rat brain was phosphorylated in vitro equally well by PKC alpha, beta, gamma and delta. Phosphoamino acid analysis indicated that both serine and threonine residues were phosphorylated. The nonphosphorylated HMG-I was shown to bind specifically to the fragment of DNA containing bp -708 to -458 of RC3 genomic DNA, which is abundant in A-T sequences. In contrast, phosphorylation of HMG-I by PKC resulted in an attenuation of binding to the DNA fragment. It is suggested that phosphorylation of HMG-I by PKC may regulate DNA binding activity of HMG-I, thereby possibly altering its biological functions.

• Emery P, Durand B, Mach B, Reith W
• RFX proteins, a novel family of DNA binding proteins conserved in the eukaryotic kingdom.
• Nucleic Acids Res. 1996; 24: 803-7
• Display abstract

• Until recently, the RFX family of DNA binding proteins consisted exclusively of four mammalian members (RFX1-RFX4) characterized by a novel highly conserved DNA binding domain. Strong conservation of this DNA binding domain precluded a precise definition of the motif required for DNA binding. In addition, the biological systems in which these RFX proteins are implicated remained obscure. The recent identification of four new RFX genes has now shed light on the evolutionary conservation of the RFX family, contributed greatly to a detailed characterization of the RFX DNA binding motif, and provided clear evidence for the function of some of the RFX proteins. RFX proteins have been conserved throughout evolution in a wide variety of species, including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans, mouse and man. The characteristic RFX DNA binding motif has been recruited into otherwise very divergent regulatory factors functioning in a diverse spectrum of unrelated systems, including regulation of the mitotic cell cycle in fission yeast, the control of the immune response in mammals, and infection by human hepatitis B virus.

• Bustin M, Reeves R
• High-mobility-group chromosomal proteins: architectural components that facilitate chromatin function.
• Prog Nucleic Acid Res Mol Biol. 1996; 54: 35-100

• Reeves R, Nissen MS
• Cell cycle regulation and functions of HMG-I(Y).
• Prog Cell Cycle Res. 1995; 1: 339-49
• Display abstract

• Members of the HMG-I(Y) family of "high mobility group" (HMG) proteins are distinguished from other nonhistone chromatin proteins by their ability to preferentially recognize the structure of the narrow minor groove of A.T-sequences of B-form DNA. In vivo the HMG-I(Y) proteins are localized in the A.T-rich G/Q bands and in the "scaffold-associated regions" (SARs) of metaphase chromosomes. These proteins also share with some of the other "HMG box" proteins the ability to recognize non-B-form structures, such as cruciforms (four-way junctions), as well as the possessing the capacity to introduce both bends and supercoils in substrate DNAs. These characteristics, along with their ability to specifically interact with a number of known transcription factors, enable the HMG-I(Y) proteins to function in vivo as structural transcription factors for a number mammalian genes. The HMG-I(Y) proteins are also in vivo substrates for the cell cycle regulated Cdc2 kinase which phosphorylates the DNA-binding domain(s) of the protein and, as a result, decreases their substrate binding affinity. This reversible in vivo pattern of Cdc2 kinase phosphorylations during the cell cycle is likely to play a major role in mediating the biological function(s) of the HMG-I(Y) proteins.

• Grasser KD
• Plant chromosomal high mobility group (HMG) proteins.
• Plant J. 1995; 7: 185-92

• Nissen MS, Reeves R
• Changes in superhelicity are introduced into closed circular DNA by binding of high mobility group protein I/Y.
• J Biol Chem. 1995; 270: 4355-60
• Display abstract

• Mammalian high mobility group HMG-I/Y chromatin proteins bind to the minor groove of A.T-rich DNA sequences with high affinity both in vivo and in vitro. Topoisomerase I-mediated relaxation assays, analyzed by one- and two-dimensional agarose gel electrophoresis, indicate that binding of recombinant human HMG-I/Y to closed circular DNA introduces positive supercoils at low protein to nucleotide molar ratios and negative supercoils at higher ratios. This is interpreted to mean that HMG-I/Y binding initially causes bending of the DNA helix followed by unwinding of the helix. In contrast, binding of another minor groove binding ligand, netropsin, introduces positive supercoils only. An in vitro produced mutant HMG-I/Y protein lacking the negatively charged carboxyl-terminal domain binds A.T-rich DNA approximately 1.4-fold better than the native protein, yet it is estimated to be 8-10-fold more effective at introducing negative supercoils. This finding suggests that the highly acidic C-terminal region of the HMG-I/Y protein may function as a regulatory domain influencing the amount of topological change induced in DNA substrates by binding of the protein. Footprinting of HMG-I/Y on negatively supercoiled A.T-rich DNA using diethylpyrocarbonate suggests that the protein is able to recognize, bind to, and alter the conformation of non-B-form DNA.

• Kim J, Reeves R, Rothman P, Boothby M
• The non-histone chromosomal protein HMG-I(Y) contributes to repression of the immunoglobulin heavy chain germ-line epsilon RNA promoter.
• Eur J Immunol. 1995; 25: 798-808
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• The rate of germ-line RNA transcription correlates with the rate of immunoglobulin heavy chain isotype switching. A promoter element for the transcription of RNA from the germ-line mouse immunoglobulin epsilon heavy chain constant region gene is induced by interleukin(IL)-4 and lipopolysaccharide, and is bound at its transcription initiation sites by an IL-4-inducible nuclear protein, NF-BRE. To examine the function of the binding site for this IL-4-inducible complex, substitution mutations were introduced in the promoter. These binding site mutations increased promoter activity and decreased binding of NF-BRE. To investigate the paradox of an IL-4-inducible protein binding to a repressor site in an IL-4-inducible promoter, we determined that the non-histone chromosomal protein HMG-I(Y) binds at the transcription initiation sites of the germ-line epsilon promoter. Assays with antisera against HMG-I(Y) revealed monomeric HMG-I(Y) in nuclear extracts. Cotransfection of an expression construct directing the synthesis of anti-sense HMG-I(Y) RNA also increased promoter activity, consistent with a repressor function of HMG-I(Y). Thus, the data are most consistent with a model in which HMG-I(Y) participates in repression of promoter activity. The effects of IL-4 may include derepression at this site.

• Wang DZ, Ray P, Boothby M
• Interleukin 4-inducible phosphorylation of HMG-I(Y) is inhibited by rapamycin.
• J Biol Chem. 1995; 270: 22924-32
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• The non-histone chromosomal protein HMG-I(Y) participates in repression of transcription directed by a promoter which confers interleukin 4 (IL-4)-inducible activation in transfected B cell lines. Metabolic labeling, phosphoamino acid analyses, and in vitro phosphorylation studies demonstrate that IL-4 induces serine phosphorylation of HMG-I(Y) in B lymphocytes. Phosphopeptide mapping shows that the predominant site of phosphorylation contains a casein kinase II consensus motif. The immunosuppressive agent rapamycin has been shown preferentially to inhibit IgE production by IL-4-treated human B cells. It is shown here that rapamycin inhibits both activation of the human germ line epsilon promoter by IL-4 and IL-4-inducible phosphorylation of HMG-I(Y). These findings demonstrate a rapamycin-sensitive pathway that transduces signals from the IL-4 receptor to nuclear factors that regulate inducible transcription. The affinity of normal nuclear HMG-I(Y) for DNA is increased by dephosphorylation in vitro, whereas in vitro kinase reactions using casein kinase II decrease recombinant HMG-I(Y) binding to DNA. These data further suggest a novel mechanism in which phosphorylation triggered by IL-4 or other cytokines could regulate the effects of HMG-I(Y) on gene transcription.

• Du W, Maniatis T
• The high mobility group protein HMG I(Y) can stimulate or inhibit DNA binding of distinct transcription factor ATF-2 isoforms.
• Proc Natl Acad Sci U S A. 1994; 91: 11318-22
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• The high mobility group protein HMG I(Y) stimulates the binding of a specific isoform of the activating transcription factor 2 (ATF-2(195)) to the interferon beta (IFN-beta) gene promoter. HMG I(Y) specifically interacts with the basic-leucine zipper region of ATF-2(195), and HMG I(Y) binds to two sites immediately flanking the ATF-2 binding site of the IFN-beta promoter. Here, we show that HMG I(Y) can stimulate the binding of ATF-2(195), at least in part, by promoting ATF-2 dimerization. In addition, we report the characterization of a naturally occurring isoform of ATF-2 (ATF-2(192)) that binds specifically to the IFN-beta promoter but is unable to interact with HMG I(Y). Remarkably, HMG I(Y) inhibits the binding of ATF-2(192) to the IFN-beta promoter. Thus, the ability of HMG I(Y) to specifically interact with ATF-2 correlates with its ability to stimulate ATF-2 binding to the IFN-beta promoter. Comparisons of the amino acid sequences of the basic-leucine zipper domains of ATF-2(195) and ATF-2(192) suggest that HMG I(Y) interacts with a short stretch of basic amino acids near the amino terminus of the basic-leucine zipper domain of ATF-2(195).

• Nieto-Sotelo J, Ichida A, Quail PH
• PF1: an A-T hook-containing DNA binding protein from rice that interacts with a functionally defined d(AT)-rich element in the oat phytochrome A3 gene promoter.
• Plant Cell. 1994; 6: 287-301
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• Phytochrome-imposed down-regulation of the expression of its own phytochrome A gene (PHYA) is one of the fastest light-induced effects on transcription reported in plants to date. Functional analysis of the oat PHYA3 promoter in a transfection assay has revealed two positive elements, PE1 and PE3, that function synergistically to support high levels of transcription in the absence of light. We have isolated a rice cDNA clone (pR4) encoding a DNA binding protein that binds to the AT-rich PE1 element. We tested the selectivity of the pR4-encoded DNA binding activity using linker substitution mutations of PE1 that are known to disrupt positive expression supported by the PHYA3 promoter in vivo. Binding to these linker substitution mutants was one to two orders of magnitude less than to the native PE1 element. Because this is the behavior expected of positive factor 1 (PF1), the presumptive nuclear transcription factor that acts in trans at the PE1 element in vivo, the data support the conclusion that the protein encoded by pR4 is in fact rice PF1. The PF1 polypeptide encoded by pR4 is 213 amino acids long and contains four repeats of the A-T hook DNA binding motif found in high-mobility group I-Y (HMGI-Y) proteins. In addition, PF1 contains an 11-amino acid-long hydrophobic region characteristic of HMG I proteins, its N-terminal region shows strong similarities to a pea H1 histone sequence and a short peptide sequence from wheat HMGa, and it shows a high degree of similarity along its entire length to the HMG Y-like protein encoded by a soybean cDNA, SB16. In vitro footprinting and quantitative gel shift analyses showed that PF1 binds preferentially to the PE1 element but also at lower affinity to two other AT-rich regions upstream of PE1. This feature is consistent with the binding characteristics of HMG I-Y proteins that are known to bind to most runs of six or more AT base pairs. Taken together, the properties of PF1 suggest that it belongs to a newly described family of nuclear proteins containing both histone H1 domains and A-T hook DNA binding domains.

• Claus P, Schulze E, Wisniewski JR
• Insect proteins homologous to mammalian high mobility group proteins I/Y (HMG I/Y). Characterization and binding to linear and four-way junction DNA.
• J Biol Chem. 1994; 269: 33042-8
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• A chromosomal high mobility group (HMG) protein from an epithelial cell line of Chironomus tentans (Diptera) was purified to homogeneity and chemically characterized. cDNA clones encoding this protein were isolated and sequenced. The deduced amino acid sequence revealed a high similarity to HMG protein I of mammalia. This insect protein has therefore been designated cHMGI. It has a deduced molecular mass of 10,371 kDa and appears to be a product of a single gene copy. Similarly to mammalian HMGI/Y proteins the insect cHMGI protein has three putative DNA-binding motifs with a sequence K/RXRGRP that are each encoded by one exon of the gene. Using synthetic peptides we have shown that the first and the second motif are necessary for high-affinity binding of the protein to DNA. Protein cHMGI binds preferentially to AT-rich DNA with a half-saturation value of 1.1 nM. Both cHMGI and human HMGI proteins recognize specifically a four-way junction DNA. We have also purified a related protein with similar physico-chemical properties from Drosophila melanogaster Kc cells. The identification and characterization of HMGI proteins in insects with polytene chromosomes and with the cytologic and genetic potential of Chironomus and Drosophila opens new possibilities for studying function(s) of this group of chromosomal proteins.

• Skalnik DG, Neufeld EJ
• Sequence-specific binding of HMG-I (Y) to the proximal promoter of the gp91-phox gene.
• Biochem Biophys Res Commun. 1993; 190: 308-9

• Balasubramanian B, Lowry CV, Zitomer RS
• The Rox1 repressor of the Saccharomyces cerevisiae hypoxic genes is a specific DNA-binding protein with a high-mobility-group motif.
• Mol Cell Biol. 1993; 13: 6071-8
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• The ROX1 gene encodes a repressor of the hypoxic functions of the yeast Saccharomyces cerevisiae. The DNA sequence of the gene was determined and found to encode a protein of 368 amino acids. The amino-terminal third of the protein contains a high-mobility-group motif characteristic of DNA-binding proteins. To determine whether the Rox1 repressor bound DNA, the gene was expressed in Escherichia coli cells as a fusion to the maltose-binding protein and this fusion was partially purified by amylose affinity chromatography. By using a gel retardation assay, both the fusion protein and Rox1 itself were found to bind specifically to a synthetic 32-bp DNA containing the hypoxic consensus sequence. We assessed the role of the general repressor Ssn6 in ANB1 repression. An ANB1-lacZ fusion was expressed constitutively in an ssn6 deletion strain, and deletion of the Rox1 binding sites in the ANB1 upstream region did not increase the level of derepression, suggesting that Ssn6 exerts its effect through Rox1. Finally, ROX1 was mapped to yeast chromosome XVI, near the ARO7-OSM2 locus.

• Skalnik DG, Neufeld EJ
• Sequence-specific binding of HMG-I(Y) to the proximal promoter of the gp91-phox gene.
• Biochem Biophys Res Commun. 1992; 187: 563-9
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• Screening of a cDNA expression library with a CCAAT-box element derived from the myelomonocyte-specific gp91-phox promoter resulted in the isolation of three independent HMG-I(Y) cDNA clones. Filter binding competition studies reveal that HMG-Y binds to this promoter element in a sequence-specific manner and exhibits a gradient of binding affinities for various A/T-rich sequences. Two adjacent A/T-rich regions within the gp91-phox promoter CCAAT-box element are required for maximal binding. In addition, competition experiments demonstrate that the binding affinity of HMG-Y is influenced by sequences that flank A/T-rich core binding sites.

• Yamaguchi-Shinozaki K, Shinozaki K
• A novel Arabidopsis DNA binding protein contains the conserved motif of HMG-box proteins.
• Nucleic Acids Res. 1992; 20: 6737-6737

• Laux T, Goldberg RB
• A plant DNA binding protein shares highly conserved sequence motifs with HMG-box proteins.
• Nucleic Acids Res. 1991; 19: 4769-4769

• Nissen MS, Langan TA, Reeves R
• Phosphorylation by cdc2 kinase modulates DNA binding activity of high mobility group I nonhistone chromatin protein.
• J Biol Chem. 1991; 266: 19945-52
• Display abstract

• Chromatin high mobility group protein I (HMG-I) is a mammalian nonhistone protein that has been demonstrated both in vitro and in vivo to preferentially bind to A.T-rich sequences of DNA. Recently the DNA-binding domain peptide that specifically mediates the in vitro interaction of high mobility group protein (HMG)-I with the narrow minor groove of A.T-DNA has been experimentally determined. Because of its predicted secondary structure, the binding domain peptide has been called "the A.T hook" motif. Previously we demonstrated that the A.T hook of murine HMG-I protein is specifically phosphorylated by purified mammalian cdc2 kinase in vitro and that the same site(s) are also phosphorylated in vivo in metaphase-arrested cells. We also found that the DNA binding affinity of short synthetic binding domain peptides phosphorylated in vitro by cdc2 kinase was significantly reduced compared with unphosphorylated peptides. Here we extend these findings to intact natural and recombinant HMG-I proteins. We report that the affinity of binding of full-length HMG-I proteins to A.T-rich sequences is highly dependent on ionic conditions and that phosphorylation of intact proteins by cdc2 kinase reduces their affinity of in vitro binding to A.T-DNA by about 20-fold when assayed near normal mammalian physiological salt concentrations. Furthermore, in cell synchronization studies, we demonstrated that murine HMG-I proteins are phosphorylated in vivo in a cell cycle-dependent manner on the same amino acid residues modified by purified cdc2 kinase in vitro. Together these results strongly support the assertion that HMG-I proteins are natural substrates for mammalian cdc2 kinase in vivo and that their cell cycle-dependent phosphorylation by this enzyme(s) significantly modulates their DNA binding affinity, thereby possibly altering their biological function(s).

• Reeves R, Langan TA, Nissen MS
• Phosphorylation of the DNA-binding domain of nonhistone high-mobility group I protein by cdc2 kinase: reduction of binding affinity.
• Proc Natl Acad Sci U S A. 1991; 88: 1671-5
• Display abstract

• Mammalian high-mobility group I nonhistone protein (HMG-I) is a DNA-binding chromatin protein that has been demonstrated both in vitro and in vivo to be localized to the A + T-rich sequences of DNA. Recently an unusual binding domain peptide, "the A.T-hook" motif, that mediates specific interaction of HMG-I with the minor groove of DNA in vitro has been described. Inspection of the A.T-hook region of the binding domain showed that it matches the consensus sequence for phosphorylation by cdc2 kinase. Here we demonstrate that HMG-I is a substrate for phosphorylation by purified mammalian cdc2 kinase in vitro. The site of phosphorylation by this enzyme is a threonine residue at the amino-terminal end of the principal binding-domain region of the protein. Labeling of mitotically blocked mouse cells with [32P]phosphate demonstrates that this same threonine residue in HMG-I is also preferentially phosphorylated in vivo. Competition binding studies show that cdc2 phosphorylation of a synthetic binding-domain peptide significantly weakens its interaction with A + T-rich DNA in vitro, and a similar weakening of DNA binding has been observed for intact murine HMG-I protein phosphorylated by the kinase in vitro. These findings indicate that cdc2 phosphorylation may significantly alter the DNA-binding properties of the HMG-I proteins. Because many cdc2 substrates are DNA-binding proteins, these results further suggest that alteration of the DNA-binding affinity of a variety of proteins is an important general component of the mechanism by which cdc2 kinase regulates cell cycle progression.

• Laux T, Seurinck J, Goldberg RB
• A soybean embryo cDNA encodes a DNA binding protein with histone and HMG-protein-like domains.
• Nucleic Acids Res. 1991; 19: 4768-4768

• Karlson JR, Mork E, Holtlund J, Laland SG, Lund T
• The amino acid sequence of the chromosomal protein HMG-Y, its relation to HMG-I and possible domains for the preferential binding of the proteins to stretches of A-T base pairs.
• Biochem Biophys Res Commun. 1989; 158: 646-51
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• The primary structure of the human high mobility group (HMG) protein HMG-Y has been established except for a few amino acids in the N-terminal and the C-terminal part of the protein. It was found that the sequence was identical to that of HMG-I except for a run of eleven amino acids. Like HMG-I the protein was N-terminally blocked and the palindromic sequence Pro-Arg-Gly-Arg-Pro occurred twice as in HMG-I. The binding of peptides derived from HMG-I (after thermolysin cleavage) to poly (dA-dT).poly(dA-dT) suggested that there are at least two different binding domains in the protein and that binding is not dependent upon an intact protein.

• Lehn DA, Elton TS, Johnson KR, Reeves R
• A conformational study of the sequence specific binding of HMG-I (Y) with the bovine interleukin-2 cDNA.
• Biochem Int. 1988; 16: 963-71
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• The DNA sequence specific interaction of the high mobility group non-histone protein HMG-I (Y) with the 3' untranslated region of the bovine interleukin-2 cDNA has been studied. Circular dichroism and thermal denaturation studies suggest that HMG-I (Y) alters the conformational state and increases the thermal stability of the DNA. Additionally, amino acid sequence analysis suggests that the previously identified non-histone protein HMG-Y is an isoform of HMG-I.

• Lund T, Dahl KH, Mork E, Holtlund J, Laland SG
• The human chromosomal protein HMG I contains two identical palindrome amino acid sequences.
• Biochem Biophys Res Commun. 1987; 146: 725-30
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• The sequence of 105 amino acids of the human high mobility group chromosomal protein HMG I has been determined. The most striking feature of this sequence is two identical palindrome sequences: pro-arg-gly-arg-pro, which together with a third related sequence: gly-arg-pro-arg, may represent the binding sites of HMG I to clusters of A-T base pairs in DNA.

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