COOPERATIVE INTERACTIONS IN DNA REPAIR

DNA 修复中的合作相互作用

• 批准号: 7034334
• 负责人: Michael G. Fried
• 金额: $ 20.7万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7034334
• 关键词: DNA repair; crosslink; enzyme activity; enzyme mechanism; methylguanine DNA methyltransferase; molecular genetics; nucleic acid structure; nucleosomes; point mutation; protein binding; protein protein interaction; stoichiometry

DESCRIPTION (provided by applicant): Our goal is to elucidate mechanisms by which DNA-repair proteins perform their essential biological functions. As immediate objectives, we will investigate the interactions of human 0 6- alkylguanine-DNA alkyltransferase (AGT) with O6-alkylguanine (lesion)-containing and lesion-free DNAs. AGT repairs pro-mutagenic O -alkylguanine residues in DNA. It binds DNA with substantial cooperativity but little sequence or base composition dependence. These results argue against mechanisms of target recognition that depend strongly on sequence. An alternate possibility, which comprises the central hypothesis of this application, is that cooperative DNA binding and access to DNA modulate the binding distributions of AGT and its rate of DNA-repair. To test this hypothesis, we will pursue three specific aims. These are: 1. To determine how binding cooperativity, supercoiling, and the presence of nucleosomes, affect the equilibrium distribution of AGT among available DNA sites and between O6-alkylguanine - containing and lesion-free sequences. 2. To identify amino acids that are present at the protein-protein interface in the cooperative AGT-DNA complex. To test the consequences of mutation of these residues on DNA binding in vitro and on DNA repair, in vitro and in vivo. 3. To identify the roles played by cooperative binding, supercoiling, and nucleosomes in the kinetic mechanisms of lesion-search by AGT and on its rate of DNA repatr. At the conclusion of this research, we will have identified the role played by cooperative binding in lesion-search, -binding and -repair, and we will have tested the notion that the rate of lesion-search depends on the structure of the DNA template. Together, these results will test the hypothesis that differences in DNA structure and accessibility determine the mechanism(s) by which AGT scans the genome for lesions and repairs them.

描述（由申请人提供）：我们的目标是阐明DNA修复蛋白执行其基本生物学功能的机制。作为直接目标，我们将研究人类0 6-烷基鸟嘌呤-DNA烷基转移酶（AGT）与O6-烷基鸟嘌呤（病变） - 含有病变的DNA的相互作用。 AGT修复DNA中的促氧化氧基O-碱基​​残基。它以实质性的合作性结合DNA，但几乎没有序列或碱基组成依赖性。这些结果反对强烈取决于序列的目标识别机制。包括该应用的中心假设的另一种可能性是，合作DNA结合和对DNA的访问调节AGT的结合分布及其DNA修复速率。为了检验这一假设，我们将追求三个具体目标。这些都是： 1。为了确定结合合作，超串联和核小体的存在如何影响可用DNA位点之间以及O6-烷基鸟嘌呤之间AGT的平衡分布 - 含有和无病变的序列。 2。确定合作AGT-DNA复合物中蛋白质蛋白界面上存在的氨基酸。为了测试这些残基在体外和DNA修复中的DNA结合中突变的后果，体外和体内。 3。确定通过AGT及其DNA repATR速率，通过合作结合，超串联和核小体在病变 - 搜索的动力学机理中扮演的角色。 在这项研究的结论中，我们将确定合作结合在病变搜索，束缚和 - 修复中所起的作用，并且我们将测试病变率率取决于DNA模板的结构的观点。总之，这些结果将检验以下假设：DNA结构和可访问性的差异决定了AGT扫描基因组的病变并修复它们的机制。