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。作为近期的目标，我们将研究人0 6-烷基鸟嘌呤-DNA烷基转移酶(AGT)与含有和无病变的O6-烷基鸟嘌呤(病变)DNA的相互作用。AGT修复DNA中具有致突变作用的O-烷基鸟嘌呤残基。它以实质上的协作性结合DNA，但很少依赖于序列或碱基组成。这些结果与强烈依赖于序列的目标识别机制相矛盾。另一种可能性，包括这一应用的中心假设，是协同DNA结合和对DNA的访问调节AGT的结合分布及其DNA修复速度。为了验证这一假设，我们将追求三个具体目标。它们是： 1.确定结合协作性、超螺旋和核小体的存在如何影响AGT在可用DNA位点之间以及在含有O6-烷基鸟嘌呤和无病变序列之间的平衡分布。 2.鉴定合作性AGT-DNA复合体中存在于蛋白质-蛋白质界面的氨基酸。检测这些残基的突变对DNA体外结合和DNA修复的影响。 3.明确协同结合、超螺旋和核小体在AGT寻找病变的动力学机制及其DNA重复率中的作用。 在这项研究的结论中，我们将确定协同结合在病变搜索、结合和修复中所起的作用，并将检验病变搜索速度取决于DNA模板结构的概念。总之，这些结果将检验这样一个假设，即DNA结构和可及性的差异决定了AGT扫描基因组是否有损伤并修复它们的机制(S)。

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.