SSB PROTEIN/DNA INTERACTIONS

SSB 蛋白质/DNA 相互作用

• 批准号: 9024307
• 负责人: Timothy M Lohman
• 金额: $ 38.13万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 2020-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9024307
• 关键词: Affect; Affinity; Amino Acids; Antibiotics; Bacteria; Bacterial Genome; Bacterial Proteins; Binding; Binding Proteins; Binding Sites; C-terminal; Cells; Chimera organism; Complex; DNA; DNA Binding; DNA Binding Domain; DNA Repair; DNA biosynthesis; DNA-Protein Interaction; Development; Disease; Equilibrium; Escherichia coli; Genetic Recombination; Genome; Goals; Homo; In Vitro; Kinetics; Knowledge; Lead; Link; Maintenance; Metabolism; Modeling; Molecular Conformation; Nucleotides; Oligonucleotides; Oligosaccharides; Organism; Peptides; Plasmodium falciparum; Play; Process; Property; Proteins; Research; Role; SS DNA BP; Single-Stranded DNA; Site; Specificity; Tail; Testing; Thermodynamics; Variant; dimer; in vivo; inhibitor/antagonist; novel; novel strategies; prototype; public health relevance; recombinational repair; single molecule; small molecule inhibitor; tool; Affect; Affinity; Amino Acids; Antibiotics; Bacteria; Bacterial Genome; Bacterial Proteins; Binding; Binding Proteins; Binding Sites; C-terminal; Cells; Chimera organism; Complex; DNA; DNA Binding; DNA Binding Domain; DNA Repair; DNA biosynthesis; DNA-Protein Interaction; Development; Disease; Equilibrium; Escherichia coli; Genetic Recombination; Genome; Goals; Homo; In Vitro; Kinetics; Knowledge; Lead; Link; Maintenance; Metabolism; Modeling; Molecular Conformation; Nucleotides; Oligonucleotides; Oligosaccharides; Organism; Peptides; Plasmodium falciparum; Play; Process; Property; Proteins; Research; Role; SS DNA BP; Single-Stranded DNA; Site; Specificity; Tail; Testing; Thermodynamics; Variant; dimer; in vivo; inhibitor/antagonist; novel; novel strategies; prototype; public health relevance; recombinational repair; single molecule; small molecule inhibitor; tool

 DESCRIPTION (provided by applicant): Single Stranded Binding (SSB) proteins are essential in all organisms due to their central roles in DNA replication, recombination and repair. Our focus is on E. coli SSB protein which is the prototypical homotetrameric bacterial SSB. E. coli SSB protein plays a central role in genome maintenance by binding single stranded (ss)DNA and by directly interacting, primarily via its 4 intrinsically disordered C- terminal tails, with a least 14 other SSB interacting proteins (SIPs) to bring them to their sites of action on DNA. Yet, how SSB selects among these many proteins (specificity) and what mechanisms these proteins use to gain access to SSB-coated ssDNA are not understood. E. coli SSB protein possesses four DNA binding domains and can bind ssDNA in multiple binding modes that differ dramatically in their properties, in particular, the number of subunits interacting with ssDNA, which affects ssDNA wrapping around the tetramer, and its inter-tetramer ssDNA binding cooperativity. The different properties of the different SSB-ssDNA binding modes can regulate access of other proteins to the ssDNA. We have recently made the surprising discovery that highly cooperative binding of SSB to ssDNA is regulated by its four intrinsically disordered C-terminal tails and have proposed a model for how these tails promote cooperativity that will be tested in the current proposal. This discovery has also allowed us to make the first SSB variant that eliminates cooperative binding and we have shown that cells expressing this variant are defective in some DNA repair processes. We will also use computational and experimental (ensemble and single molecule) approaches to examine how the amino acid composition of the intrinsically disordered linker region of the C-terminal tails affects its conformational states an cooperativity. We have also developed novel approaches to make SSB variants that can selectively populate the different SSB-ssDNA binding modes. These SSB variants will be used to determine whether either of the major binding modes is dispensable in E. coli and what processes might be affected if SSB is restricted from accessing that particular mode. Ensemble thermodynamic approaches will also be used to investigate a novel allosteric effect of the SSB C-terminal tails on binding of at least one SIP to ssDNA. E. coli SSB is an essential bacterial protein and is involved in all DNA metabolic processes through its interactions with ssDNA and other proteins (SIPs). These SSB-SIP interactions provide a novel target for potentially novel antibiotics and our basic studies of EcoSSB and its C-terminal tails can facilitate the search and development of such novel antibiotics.

 描述（由应用提供）：由于其在DNA复制，重组和修复中的主要作用，因此在所有生物体中，单链结合（SSB）蛋白都是必不可少的。我们的重点是大肠杆菌SSB蛋白，该蛋白是典型的同型细菌SSB。大肠杆菌SSB蛋白通过结合单链（SS）DNA和直接相互作用（通过其4种本质上无序的C-末端尾巴）与至少14个其他SSB相互作用蛋白（SIP）（SIPS）通过其主要相互作用，在基因组维持中起着核心作用。但是，在这些众多蛋白质（特异性）中如何选择SSB以及这些蛋白质用于访问SSB涂层ssDNA的机制。大肠杆菌SSB蛋白具有四个DNA结合结构域，并且可以在多种结合模式中结合ssDNA，这些结合模式在其性质中截然不同，特别是与ssDNA相互作用的亚基数量，这会影响ssDNA围绕四聚体及其跨四分机及其跨tetramer ssDNA结合均衡。不同SSB-SSDNA结合模式的不同特性可以调节其他蛋白质对ssDNA的访问。我们最近提出了一个令人惊讶的发现，SSB与SSDNA的高度协调结合受其四个本质上无序的C末端尾巴的调节，并提出了一个模型，以促进这些尾巴如何促进当前建议中测试的协调。这一发现还使我们能够制造出消除合作结合的第一个SSB变体，我们已经表明，表达该变体的细胞在某些DNA修复过程中有缺陷。我们还将使用计算和实验性（集合和单分子）方法来检查C末端尾巴本质上无序接头区域的氨基酸组成如何影响其构象状态的配位状态。我们还开发了新的方法来制造可以选择性地填充不同SSB-SSDNA结合模式的SSB变体。这些SSB变体将用于确定大肠杆菌中的任何一种主要结合模式是否可以分配，如果SSB受到限制访问该特定模式，则可能会影响哪些过程。集合热力学方法还将用于研究SSB C末端尾巴对至少一种SIP与SSDNA结合的新型变构作用。大肠杆菌SSB是一种必不可少的细菌蛋白，通过其与ssDNA和其他蛋白质（SIPS）的相互作用参与了所有DNA代谢过程。这些SSB-SIP相互作用为潜在的新型抗生素提供了新的靶标，而我们对ECOSSB及其C末端尾巴的基础研究可以促进这种新型抗生素的搜索和开发。