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，也与散发性肿瘤的病因学有关。由于DNA复制错误、非法重组事件和对某些DNA损伤剂（包括一些用作抗肿瘤药物）的正常反应失败，该途径的失活使细胞在遗传上不稳定。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。虽然有七种活性与人类链特异性错配修复有关（MutS_, MutS_, MutLo_， PCNA， RPA， EXOI和DNA聚合酶$），但这些不足以重建反应。所要求的该项目的延长的一个主要目标是确定和隔离其他所需的活动。MutSo_和MutL_在错配修复的启动中起着关键作用，但它们与DNA相互作用的分子模式及其核苷酸水解功能的作用尚未明确，并且一直是争议的主题。本应用程序的第二个目的是解决这些问题，重点关注与DNA相互作用有关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。在错配修复反应的早期阶段涉及的多蛋白质和多蛋白质- dna组装的性质也将被检查。这些研究将着重于MutSo_与MutLo_的相互作用，MutSo_与PCNA的相互作用，以及EXOI与MutSo_和MutLa的相互作用，以及RPA对活化的EXOI的调节。第四行提出的工作解决了错配修复系统和BLM解旋酶在重组保真度中的作用。在错配修复缺陷和BLM缺陷细胞中，非法重组事件增加，并且MutSo_已被证明可以调节BLM解旋酶的活性。因此，我们将询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。网站性能 ======================================== 节结束 ===========================================

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===========================================