COOPERATIVE INTERACTIONS IN DNA REPAIR

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。作为近期目标，我们将研究人0 6-烷基鸟嘌呤-DNA烷基转移酶（AGT）与含0 6-烷基鸟嘌呤（病变）和无病变DNA的相互作用。AGT修复DNA中的前致突变O -烷基鸟嘌呤残基。它与DNA的结合具有很大的协同性，但对序列或碱基组成的依赖性很小。这些结果反对强烈依赖于序列的靶识别机制。另一种可能性，其中包括本申请的中心假设，是合作DNA结合和访问DNA调节AGT的结合分布和DNA修复的速度。为了验证这一假设，我们将追求三个具体目标。这些措施是： 1.确定结合协同性、超螺旋和核小体的存在如何影响AGT在可用DNA位点之间以及含O 6-烷基鸟嘌呤和无损伤序列之间的平衡分布。 2.鉴定AGT-DNA协同复合物中存在于蛋白质-蛋白质界面的氨基酸。测试这些残基突变对体外DNA结合以及体外和体内DNA修复的影响。 3.探讨协同结合、超螺旋和核小体在AGT损伤搜索动力学机制中的作用及其对DNA修复率的影响。 在这项研究的结论，我们将确定合作绑定在病变搜索，结合和修复中发挥的作用，我们将测试的概念，病变搜索的速度取决于DNA模板的结构。总之，这些结果将检验DNA结构和可及性的差异决定AGT扫描基因组病变并修复它们的机制的假设。

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.