Enzymology Of Eukaryotic Mismatch Repair

错配修复是遗传稳定性的主要贡献者。这种途径具有临床意义，因为 错配修复基因的突变是遗传性结肠炎最常见的形式之一的原因。 癌症，并已牵连到一个子集（约15%）的散发性肿瘤的发展，可以发生 在各种组织中。错配修复缺陷也对癌症治疗有影响， 该途径的失活使细胞对某些抗肿瘤药物的细胞毒性作用具有抗性。的 该项目的主要目标是阐明这种DNA修复系统的分子性质，因为它在 复制错误纠正和细胞对DNA损伤的反应。我们的目标有四个方面：（1）9 活性（MutSa，MutS（3，MutLa，Exol，RFC，PCNA，RPA，HMGB 1和DNA聚合酶6）已被 与真核细胞错配修复有关，但很明显， 反应中的重要调节作用。我们打算将这些活动分离出来， 反应。(2)我们已经证明，已知的人类错配修复活动的子集催化 几个部分反应，至少部分地解释了整个反应的复杂性。我们计划 进一步表征相应的多蛋白质DNA组装体的分子生物学特性， 活性，化学计量和构象，以及腺嘌呤核苷酸对这些的调节作用， 参数(3)人类识别和处理错配/DNA损伤的结构基础 失配修复系统将在与洛雷纳Beese的合作X射线晶体学研究中得到解决 实验室(4)MutSa和MutLa缺陷使哺乳动物细胞对SN 1的细胞毒性作用具有抗性 DNA甲基化剂，6-硫鸟嘌呤和顺铂。为了阐明人类基因错配的功能 修复系统在DNA损伤反应中的作用，我们将继续进行体外实验，希望能阐明 ATR损伤信号激酶的MutSa和MutLa依赖性激活机制 O 6-甲基鸟嘌呤损伤

Mismatch repair is a major contributor to genetic stability. This pathway is of clinical interest because mutations in mismatch repair genes are the cause of one of the most prevalent forms of hereditary colon cancer, and have been implicated in the development of a subset (¿ 15%) of sporadic tumors that can occur in a variety of tissues. Mismatch repair defects also have implications for cancer treatment because inactivation of the pathway renders cells resistant to the cytotoxic effects of certain anti-tumor drugs. The primary goals of this project are to elucidate the molecular nature of this DNA repair system as it functions in replication error correction and in the cellular response to DNA damage. Our aims are four-fold: (1) Nine activities (MutSa, MutS(3, MutLa, Exol, RFC, PCNA, RPA, HMGB1, and DNA polymerase 6) have been implicated in eukaryotic mismatch repair, but it is clear that additional, and as yet unknown, components play important regulatory roles in the reaction. We intend to isolate these activities and establish their functions in the reaction. (2) We have shown that subsets of the known human mismatch repair activities catalyze several partial reactions that account at least in part for the complexity of the overall reaction. We plan further characterization of the corresponding multi-protein¿DNA assemblies with respect to molecular activities, stoichiometry, and conformation, as well as modulatory effects of adenine nucleotides on these parameters. (3) The structural basis of mismatch/DNA lesion recognition and processing by the human mismatch repair system will be addressed in collaborative X-ray crystallographic studies with Lorena Beese's laboratory. (4) MutSa and MutLa defects render mammalian cells resistant to the cytotoxic effects of SN1 DNA methylators, 6-thioguanine, and cisplatin. In an attempt to clarify the function of the human mismatch repair system in the DNA damage response, we will pursue in vitro experiments with the hope of elucidating the mechanism of MutSa- and MutLa-dependent activation of the ATR damage-signaling kinase in response to O6-methylguanine lesions.