Mismatch Repair Interactions

DNA错配修复是遗传稳定性的主要因素。错配修复缺陷赋予强烈的癌症易感性，并对癌症治疗产生影响，因为该途径的失活使细胞抵抗某些抗肿瘤药物的细胞毒性作用，这是该系统参与DNA损伤反应的结果。也许令人惊讶的是，错配修复功能对于某些突变的产生也是必需的，例如（CAG）{n}重复序列的扩增，这是许多神经退行性疾病的主要原因。该项目的目标是阐明多蛋白、蛋白质-DNA和多蛋白DNA组装的构象、构象变异和结构，这些组装是错配修复系统中DNA损伤处理和损伤信号传导的关键中间体。我们的目标有三个方面：(1)使用氘交换质谱（DXMS），我们已经确定了细菌MutS和真核MutS- α (MSH2-MSH6)的区域，这些区域经历了依赖底物的构象转变。这些区域将遭受定点诱变和由此产生的突变，其特征在于它们对体内和体外错配修复的影响。(2)小角度x射线散射、平衡方法、DXMS、x射线晶体学、生化和遗传学方法将被适当地用于扩展我们对错配修复中涉及的多蛋白质组装的理解。这些组合将包括与mts - α (MSH2-MSH6)和mutl - α (MLH1-PMS1/PMS2)的PCNA复合物，与外切酶1和Chk1的MutSa复合物，以及外切酶1和BLM解旋酶之间的复合物。后一项研究将与项目4合作进行。(3)人类muts - α和muts - β与PCNA和mutl - α相互作用的方式不同，表明muts - α和muts - β触发的修复事件是通过明显不同的机制进行的。鉴于已知MutS- β和mutl - α参与体细胞期（CAG）{n}:(CTG){n}，三重重复扩增，我们将通过筛选选择性阻断mutl - α -MutS- β - dna三元复合物组装的化合物来寻找特异性阻断MutS- β触发的修复事件的小分子抑制剂。

DNA mismatch repair is a major contributor to genetic stability. Mismatch repair defects confer strong cancer predisposition and have implications for cancer therapy because inactivation of the pathway renders cells resistant to the cytotoxic effects of certain anti-tumor drugs, a consequence of participation of the system in the DNA damage response. Perhaps surprisingly, mismatch repair function is also required for production of certain mutations, such as the expansion of (CAG){n} repeat sequences, the primary cause of a number of neurodegenerative diseases. The goals of this project to clarify conformations, conformational variation, and structures of multi-protein, protein-DNA, and multi-protein DNA assemblies that are key intermediates in DNA lesion processing and damage signaling by the mismatch repair system. Our aims are 3-fold: (1) Using deuterium exchange mass spectrometry (DXMS), we have identified regions of bacterial MutS and eukaryotic MutS-alpha (MSH2-MSH6) that undergo substrate-dependent conformational transitions. These regions will be subjected to site-directed mutagenesis and the resulting mutants characterized for their impact on mismatch repair both in vivo and in vitro. (2) Small angle X-ray scattering, equilibrium methods, DXMS, x-ray crystallographic, biochemical and genetic approaches as appropriate will be utilized to extend our understanding of multi-protein assemblies involved in mismatch repair. These assemblies will include PCNA complexes with MutS-alpha (MSH2-MSH6) and MutL-alpha (MLH1-PMS1/PMS2), MutSa complexes with exonuclease 1 and with Chk1, and the complex between exonuclease 1 and the BLM helicase. The latter study will be pursued in collaboration with Project 4. (3) Human MutS-alpha and MutS-Beta differ in the manner in which they interact with PCNA and MutL-alpha, indicating that MutS-alpha- and MutS-Beta-triggered repair events proceed by significantly different mechanisms. In view of the known involvement of MutS-Beta and MutL-alpha in the somatic phase (CAG){n}:(CTG){n}, triplet repeat expansion, we will seek small molecule inhibitors that specifically block MutS Beta-triggered repair events by screening for compounds that selectively block assembly the MutL-alpha-MutS-Beta-DNA ternary complex.