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Multiple biochemical activities of NM23/NDP kinase in gene regulation 总被引:11,自引:0,他引:11
Postel EH 《Journal of bioenergetics and biomembranes》2003,35(1):31-40
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The V(D)J recombination signal sequence and kappa B binding protein Rc binds DNA as dimers and forms multimeric structures with its DNA ligands. 总被引:1,自引:0,他引:1
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The murine DNA binding protein Rc binds to the heptamer motif of the V(D)J recombination signal sequences and to the kappa B motif of the immunoglobulin enhancer. Bacterial fusion proteins for Rc and DNA ligands of Rc form multiple protein-DNA complexes in electrophoretic mobility shift assays (EMSA). Large complexes formation is favored by an increased Rc concentration. In order to determine the architecture of these complexes, the apparent molecular weights of the protein-DNA complexes were first determined by their gel mobilities. The data suggest that Rc binds to its DNA ligands as dimers, tetramers, and multiples of tetramers. The inference that Rc binds DNA as dimers was substantiated by the formation of chimeric complexes when two electrophoretically distinguishable Rc proteins were employed in EMSA. Methylation interference experiments show that there are no contiguous protein binding sites evident in the DNA of the larger complexes. Apparently, multimerization occurs via protein-protein interactions. Such interaction was demonstrated by the formation of Rc dimers and tetramers in a chemical crosslinking experiment. Significantly, the multimerization of DNA-bound Rc could be involved in bringing the variable region gene segments together for the somatic V(D)J recombination. 相似文献
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C P Verrijzer J A van Oosterhout W W van Weperen P C van der Vliet 《The EMBO journal》1991,10(10):3007-3014
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A detailed analysis of the DNA-binding sites of 26 proteins is presented using data from the Nucleic Acid Database (NDB) and the Protein Data Bank (PDB). Chemical and physical properties of the protein-DNA interface, such as polarity, size, shape, and packing, were analysed. The DNA-binding sites shared common features, comprising many discontinuous sequence segments forming hydrophilic surfaces capable of direct and water-mediated hydrogen bonds. These interface sites were compared to those of protein-protein binding sites, revealing them to be more polar, with many more intermolecular hydrogen bonds and buried water molecules than the protein-protein interface sites. By looking at the number and positioning of protein residue-DNA base interactions in a series of interaction footprints, three modes of DNA binding were identified (single-headed, double-headed and enveloping). Six of the eight enzymes in the data set bound in the enveloping mode, with the protein presenting a large interface area effectively wrapped around the DNA.A comparison of structural parameters of the DNA revealed that some values for the bound DNA (including twist, slide and roll) were intermediate of those observed for the unbound B-DNA and A-DNA. The distortion of bound DNA was evaluated by calculating a root-mean-square deviation on fitting to a canonical B-DNA structure. Major distortions were commonly caused by specific kinks in the DNA sequence, some resulting in the overall bending of the helix. The helix bending affected the dimensions of the grooves in the DNA, allowing the binding of protein elements that would otherwise be unable to make contact. From this structural analysis a preliminary set of rules that govern the bending of the DNA in protein-DNA complexes, are proposed. 相似文献
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A method is presented to predict overall conformations of protein-DNA complexes on the basis of the known three-dimensional structures of the proteins. The method is restricted to proteins with a common twofold symmetry axis, which show only minor conformational changes upon binding to DNA. The method uses a numerical finite difference solution of the linearized Poisson-Boltzmann equation and subsequent energy minimization cycles. Structural parameters—the rotation angle of the DNA relative to the protein around the common symmetry axis, the protein-DNA distance, and intermolecular hydrogen-bonding contacts—are presented for two test cases, DNA bound to CAP (catabolite gene activator protein) and to the Cro-repressor of bacteriophage 434. The DNA curvature in the starting model of the docking procedure was chosen as a smoothed approximation of the conformation found in the X-ray structures of these complexes. The method is further used to predict the unknown structure of the complex between the factor for inversion stimulation (FIS) and DNA, which is bent upon binding to FIS. In contrast to the test cases, the unknown curvature of the starting model is derived from a calibration of electrostatic precalculations for different proteins according to crystallographically observed DNA bending. The results of the modeling are in good accordance with the experimentally observed overall structure of protein-DNA complexes for the two test cases; for FIS, they correspond to several of the experimentally proposed protein-DNA contacts. © 1996 Wiley-Liss, Inc. 相似文献
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The ubiquitous, eukaryotic, high-mobility group box (HMGB) chromosomal proteins promote many chromatin-mediated cellular activities through their non-sequence-specific binding and bending of DNA. Minor-groove DNA binding by the HMG box results in substantial DNA bending toward the major groove owing to electrostatic interactions, shape complementarity, and DNA intercalation that occurs at two sites. Here, the structures of the complexes formed with DNA by a partially DNA intercalation-deficient mutant of Drosophila melanogaster HMGD have been determined by X-ray crystallography at a resolution of 2.85 Å. The six proteins and 50 bp of DNA in the crystal structure revealed a variety of bound conformations. All of the proteins bound in the minor groove, bridging DNA molecules, presumably because these DNA regions are easily deformed. The loss of the primary site of DNA intercalation decreased overall DNA bending and shape complementarity. However, DNA bending at the secondary site of intercalation was retained and most protein-DNA contacts were preserved. The mode of binding resembles the HMGB1 box A-cisplatin-DNA complex, which also lacks a primary intercalating residue. This study provides new insights into the binding mechanisms used by HMG boxes to recognize varied DNA structures and sequences as well as modulate DNA structure and DNA bending. 相似文献
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Rodney E. Harrington 《Molecular microbiology》1992,6(18):2549-2555
Most biological events are regulated at the molecular level by site-specific associations between specialized proteins and DNA. These associations may bring distal regions of the genome into functional contact or may lead to the formation of large multisubunit complexes capable of regulating highly site-specific transactional events. It is now believed that sequence-specific protein-DNA recognition and the ability of certain proteins to compete for multiple binding sites is regulated at several levels by the local structure and conformation of the binding partners. These encompass the microstructure of DNA, including its curvature, bending and flexing as well as conformational lability in the DNA-binding domains of the proteins. Possible mechanisms for binding specificity are discussed in the context of specific nucleoprotein systems with particular emphasis given to the roles of DNA conformations in these interactions. 相似文献
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Swanson PC 《Molecular and cellular biology》2002,22(5):1340-1351
Two lymphoid cell-specific proteins, RAG-1 and RAG-2, initiate V(D)J recombination by introducing DNA breaks at recombination signal sequences (RSSs). Although the RAG proteins themselves bind and cleave DNA substrates containing either a 12-RSS or a 23-RSS, DNA-bending proteins HMG-1 and HMG-2 are known to promote these processes, particularly with 23-RSS substrates. Using in-gel cleavage assays and DNA footprinting techniques, I analyzed the catalytic activity and protein-DNA contacts in discrete 12-RSS and 23-RSS complexes containing the RAG proteins and either HMG-1 or HMG-2. I found that both the cleavage activity and the pattern of protein-DNA contacts in RAG-HMG complexes assembled on 12-RSS substrates closely resembled those obtained from analogous 12-RSS complexes lacking HMG protein. In contrast, 23-RSS complexes containing both RAG proteins and either HMG-1 or HMG-2 exhibited enhanced cleavage activity and displayed an altered distribution of cleavage products compared to 23-RSS complexes containing only RAG-1 and RAG-2. Moreover, HMG-dependent heptamer contacts in 23-RSS complexes were observed. The protein-DNA contacts in RAG-RSS-HMG complexes assembled on 12-RSS or 23-RSS substrates were strikingly similar at comparable positions, suggesting that the RAG proteins mediate HMG-dependent heptamer contacts in 23-RSS complexes. Results of ethylation interference experiments suggest that the HMG protein is positioned 5' of the nonamer in 23-RSS complexes, interacting largely with the side of the duplex opposite the one contacting the RAG proteins. Thus, HMG protein plays the dual role of bringing critical elements of the 23-RSS heptamer into the same phase as the 12-RSS to promote RAG binding and assisting in the catalysis of 23-RSS cleavage. 相似文献
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The β recombinase is unable to mediate in vitro DNA recombination between two directly oriented recombination sites unless a bacterial chromatin-associated protein ( Bacillus subtilis Hbsu or Eschrichia coli HU) is provided. By electron microscopy, we show that the role of Hbsu is to help in joining the recombination sites to form a stable synaptic complex. Some evidence supports the fact that Hbsu works by recognizing and stabilizing a DNA structure at the recombination site, rather than by serving as a bridge between β recombinase dimers through a protein-protein interaction. We show that the mammalian HMG1 protein, which shares neither sequence nor structural homology with Hbsu, can also stimulate β-mediated recombination. These chromatin-associated proteins share the property of binding to DNA in a relatively non-specific fashion, bending it, and having a marked preference for altered DNA structures. Hbsu, HU or HMG1 proteins probably bind specifically at the crossing-over region, since at limiting protein-DNA molar ratios they could not be outcompeted by an excess of a DNA lacking the crossing over site. Distamycin, a minor groove binder that induces local distortions in DNA, did not affect the binding of β protein to DNA, but inhibited the formation of the synaptic complex. 相似文献
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Defects in chromosome replication can lead to translocations that are thought to result from recombination events at stalled DNA replication forks. The progression of forks is controlled by an essential DNA helicase, which unwinds the parental duplex and can stall on encountering tight protein-DNA complexes. Such pause sites are hotspots for recombination and it has been proposed that stalled replisomes disassemble, leading to fork collapse. However, in both prokaryotes and eukaryotes it now seems that paused forks are surprisingly stable, so that DNA synthesis can resume without recombination if the barrier protein is removed. Recombination at stalled forks might require other events that occur after pausing, or might be dependent on features of the surrounding DNA sequence. These findings have important implications for our understanding of the regulation of genome stability in eukaryotic cells, in which pausing of forks is mediated by specific proteins that are associated with the replicative helicase. 相似文献
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Processing of recombination intermediates in vitro 总被引:6,自引:0,他引:6
S C West 《BioEssays : news and reviews in molecular, cellular and developmental biology》1990,12(4):151-154
Genetic recombination involves the exchange of genetic material between chromosomes to produce new assortments of alleles. As such, it affects one of the most fundamental and important components of heredity--the genome itself. To understand the molecular basis of recombination, efforts have been directed to try to determine how simple organisms recombine their DNA. One approach involves the development of in vitro systems in which recombination reactions can be studied using purified enzymes. Detailed studies of these systems, using enzymes isolated from bacteria and bacterial viruses, indicate the formation of unique protein-DNA complexes. The structure of the DNA within these complexes has important consequences for the subsequent formation of recombinant products. 相似文献
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Michael Gromiha M Siebers JG Selvaraj S Kono H Sarai A 《Journal of molecular biology》2004,337(2):285-294
Protein-DNA recognition plays an essential role in the regulation of gene expression. Regulatory proteins are known to recognize specific DNA sequences directly through atomic contacts (intermolecular readout) and/or indirectly through the conformational properties of the DNA (intramolecular readout). However, little is known about the respective contributions made by these so-called direct and indirect readout mechanisms. We addressed this question by making use of information extracted from a structural database containing many protein-DNA complexes. We quantified the specificity of intermolecular (direct) readout by statistical analysis of base-amino acid interactions within protein-DNA complexes. The specificity of the intramolecular (indirect) readout due to DNA was quantified by statistical analysis of the sequence-dependent DNA conformation. Systematic comparison of these specificities in a large number of protein-DNA complexes revealed that both intermolecular and intramolecular readouts contribute to the specificity of protein-DNA recognition, and that their relative contributions vary depending upon the protein-DNA complexes. We demonstrated that combination of the intermolecular and intramolecular energies derived from the statistical analyses lead to enhanced specificity, and that the combined energy could explain experimental data on binding affinity changes caused by base mutations. These results provided new insight into the relationship between specificity and structure in the process of protein-DNA recognition, which would lead to prediction of specific protein-DNA binding sites. 相似文献
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New approaches to chromatin function 总被引:12,自引:0,他引:12
A P Wolffe 《The New biologist》1990,2(3):211-218
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