Mismatch Repair Interactions

DNA错配修复是遗传稳定性的主要因素。错配修复缺陷具有强烈的癌症易感性，并对癌症治疗产生影响，因为该途径的失活使细胞对某些抗肿瘤药物的细胞毒作用产生抗药性，这是该系统参与DNA损伤反应的结果。也许令人惊讶的是，错配修复功能也是产生某些突变所必需的，例如(CAG){n}重复序列的扩展，这是许多神经退行性疾病的主要原因。本项目的目标是阐明多蛋白质、蛋白质-DNA和多蛋白质DNA组件的构象、构象变化和结构，这些组件是错配修复系统在DNA损伤处理和损伤信号传递中的关键中间体。我们的目标有三个方面：(1)利用氢交换质谱仪(DXMS)，我们已经确定了细菌MutS和真核生物MutS-α(MSH2-MSH6)经历底物依赖构象转变的区域。这些区域将受到定点突变，由此产生的突变体在体内和体外都以其对错配修复的影响为特征。(2)适当地利用小角X射线散射、平衡方法、DXMS、X射线结晶学、生化和遗传学方法来扩大我们对错配修复中涉及的多蛋白质组合的理解。这些组件包括增殖细胞核抗原与MutS-α(MSH2-MSH6)和MutL-α(MLH1-PMS1/PMS2)的复合体，MUTSA与外核酸酶1和Chk1的复合体，以及外核酸酶1和BLM解旋酶之间的复合体。后一项研究将与项目4合作进行。(3)人类MutS-α和MutS-Beta与增殖细胞核抗原和MutL-α相互作用的方式不同，表明MutS-α和MutS-Beta触发的修复事件通过显著不同的机制进行。鉴于已知的MutS-Beta和MutL-α参与了体细胞相(CAG){n}：(CTG){n}，三联体重复序列的扩展，我们将通过筛选选择性阻断MutL-α-MutS-Beta-DNA三元复合体组装的化合物来寻找特异性阻断MutS Beta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.