Enzymology and Eukaryotic Mismatch Repair

超出所提供的空间。人类错配修复系统的缺陷是遗传性非息肉病性结肠癌和家族性结肠癌的原因，并且还与散发性肿瘤的病因学有关。由于DNA复制错误、非法重组事件和不能正常响应某些DNA损伤剂（包括用作抗肿瘤药物的几种），该途径的失活使细胞遗传上不稳定。尽管这种遗传稳定系统的重要性，我们对其分子性质的理解是有限的。尽管在人链特异性错配修复中涉及七种活性（MutS_、MutS_、MutLo_、PCNA、RPA、EXOI和DNA聚合酶$），但这些不足以重建反应。请求延长该项目的一个主要目标是确定和分离其他必要活动。MutSo_和MutL_在错配修复的启动中起关键作用，但是它们与DNA相互作用的分子模式以及它们的核苷酸水解功能的作用还没有很好地定义，并且一直是争议的主题。本申请的第二个目的是解决这些问题，重点是与DNA相互作用中涉及的蛋白质寡聚化状态，并进一步阐明其ATP酶中心作为DNA-蛋白质相互作用调节剂的功能。还将研究错配修复反应早期阶段涉及的多蛋白质和多蛋白质-DNA组装体的性质。这些研究将强调MutSo_与MutLo_、MutS_与PCNA以及EXOI与MutSo_和MutLa的相互作用，以及RPA对激活的EXOI的调节。第四行提出的工作解决的错配修复系统和BLM解旋酶在重组保真度的作用。在错配修复缺陷型和BLM缺陷型细胞中，不合理重组事件增加，并且MutSo_已显示调节BLM解旋酶的活性。因此，我们将问这两个系统是否以协调的方式发挥作用以抑制非法重组事件。性能现场=

EXCEED THE SPACE PROVIDED. Defects in the human mismatch repair system are the cause of hereditary nonpolyposis colon cancer and familial colon cancer, and have also been implicated in etiology of sporadic tumors. Inactivation of this pathway renders cells genetically unstable due to DNA replication errors, illegitimate recombination events, and failure to respond normally to certain DNA damaging agents, including several that are used as anti- tumor drugs. Despite the importance of this genetic stabilization system, our understanding of its molecular nature is limited. Although seven activities have been implicated in human strand-specific mismatch repair (MutS_, MutS_, MutLo_, PCNA, RPA, EXOI, and DNA polymerase $), these are not sufficient to reconstitute the reaction. A major goal for the requested extension of this project is the identification and isolation of other required activities. MutSo_ and MutL_ play key roles in the initiation of mismatch repair, but the molecular modes by which they interact with DNA and the roles of their nucleotide hydrolytic functions are not well defined and have been the subject of controversy. The second aim of this application addresses these questions, with emphasis on the protein oligomerization states involved in the interaction with DNA, and further clarification of the function of their ATPase centers as modulators of DNA-protein interaction. The nature of multi-protein and multi-protein-DNA assemblies involved in early stages of the mismatch repair reaction will also be examined. These studies will emphasize interactions of MutSo_ with MutLo_, MutS(_ with PCNA, and EXOI with MutSo_ and MutLa, as well as modulation of activated EXOI by RPA. The fourth line of proposed work addresses the role of the mismatch repair system and the BLM helicase in recombination fidelity. Illegitimate recombination events are elevated in mismatch repair-deficient and in BLM-deficient cells, and MutSo_ has been shown to modulate the activity of the BLM helicase. Consequently, we will ask whether these two systems function in a coordinated manner to suppress illegitimate recombination events. PERFORMANCE SITE ========================================Section End===========================================