Enzymology Of Eukaryotic Mismatch Repair

错配修复是遗传稳定性的主要因素。这一途径具有临床意义，因为 错配修复基因突变是一种最常见的遗传性结肠疾病的原因 癌症，并与可能发生的散发性肿瘤的亚群(15%)的发展有关 在各种组织中。错配修复缺陷也对癌症治疗有影响，因为 该途径的失活使细胞对某些抗肿瘤药物的细胞毒作用产生抗药性。这个 这个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：(一)九个 活性(MutSA、MutS(3，Mutla、Exol、RFC、PCNA、RPA、HMGB1和DNA聚合酶6) 与真核细胞错配修复有关，但很明显，其他未知的成分在其中发挥作用 在反应中起着重要的调节作用。我们打算将这些活动分离出来，并在 这种反应。(2)我们已经证明了已知的人类错配修复活动的子集催化 几个部分反应，至少部分解释了整个反应的复杂性。我们计划 与分子相关的相应多蛋白DNA组件的进一步表征 活性、化学计量学和构象，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 错配修复系统将在与Lorena Beese的合作X射线晶体研究中得到解决 实验室。(4)MUTSA和MUTRA缺陷使哺乳动物细胞对SN1的细胞毒作用产生抵抗 DNA甲基化体、6-硫代鸟嘌呤和顺铂。试图澄清人类错配的功能 在DNA损伤修复系统的反应中，我们将进行体外实验，希望能阐明 依赖MUTSA和MUTRA激活ATR损伤信号通路的机制 到O6-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.