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0 6-烷基鸟嘌呤（病变）和无病变dna的相互作用。AGT修复DNA中致突变的O -烷基鸟嘌呤残基。它结合DNA具有很大的协同性，但很少依赖于序列或碱基组成。这些结果反驳了强烈依赖序列的目标识别机制。另一种可能性，包括本应用的中心假设，是DNA的合作结合和对DNA的访问调节了AGT的结合分布及其DNA修复率。为了验证这一假设，我们将追求三个具体目标。这些都是:

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.