Mechanistic studies of DNA repair and damage response

该提案的总体目标是阐明MutS的结构和动力学特性， DNA错配修复（MMR）启动信号与DNA错配修复（MMR）启动信号的DNA复合物 损伤诱导的凋亡。MMR是纠正DNA合成错误的机制 在复制后，它是维持基因组完整性的核心。MMR 蛋白质还参与其他几种DNA处理，包括DNA损伤传感。在 除了显著增加突变频率外，MMR基因的失活 减少细胞凋亡，增加细胞存活，并导致对化疗的抗性。在人类中， MMR基因突变与遗传性非息肉病性结直肠癌直接相关， 与几种散发性癌症有关。MMR是由MutS同源物结合到一个 不匹配.随后，MutL同源物与MutS同源物以ATP依赖性相互作用。 方式和协调蛋白质-蛋白质相互作用，信号切除和再合成的 含有错误核苷酸的新合成DNA链。启动DNA损伤- 真核生物中诱导的凋亡遵循非常相似的起始途径。MutS同系物 MSH 2-MSH 6（MutS）优先与DNA损伤结合，随后与 MutL同源物MLH 1-PMS 2（酵母中的MLH 1-PMS 1; MutL？），但不是起始DNA 在错配修复中，这些相互作用激活细胞周期检查点并发出凋亡信号。的 这项资助的中心目标是了解MutS与受损DNA的相互作用如何发出信号， 细胞周期检查点反应和凋亡，而MutS <$与错配的相互作用 信号MMR。我们试图揭示MutS <$-DNA的构象和动力学性质 复合物引发不同的细胞命运取决于MutS？是否与错配结合 或者DNA损伤我们提出了一个系统的一系列实验中，我们使用一个 结合体和单分子荧光技术与原子荧光 力显微镜来表征MutS的结合和动态构象特性， DNA复合物，信号修复与凋亡。我们将研究野生型 和MutS的突变体，其表现出MMR和DNA损伤的功能分离 反应，与受损的DNA和与表现出不同的修复效率的错配， 核苷酸辅因子的存在和不存在。

The overall goal of this proposal is to elucidate the structural and dynamic properties of MutS¿- DNA complexes that signal initiation of DNA mismatch repair (MMR) versus initiation of DNA damage-induced apoptosis. MMR is the mechanism by which DNA synthesis errors are corrected post-replicatively, and it is central to the maintenance to the integrity of the genome. MMR proteins are also involved in several other DNA transactions, including DNA damage sensing. In addition to dramatically increasing the frequency of mutations, inactivation of MMR genes decreases apoptosis, increases cell survival, and results in resistance to chemotherapy. In humans, mutations in MMR genes are directly linked to hereditary non-polyposis colorectal cancer and are associated with several sporadic cancers. MMR is initiated by MutS homologs binding to a mismatch. Subsequently, MutL homologs interact with the MutS homologs in an ATP dependent manner and coordinate protein-protein interactions that signal excision and resynthesis of the newly synthesized DNA strand containing the incorrect nucleotide. Initiation of DNA damage- induced apoptosis in eukaryotes follows a very similar initiation pathway. The MutS homolog MSH2-MSH6 (MutS¿) binds preferentially to a DNA lesion and subsequently interacts with the MutL homolog MLH1-PMS2 (MLH1-PMS1 in yeast; MutL¿), but instead of initiating DNA mismatch repair, these interactions activate cell-cycle checkpoints and signal apoptosis. The central goal of this grant is to understand how interaction of MutS¿ with damaged DNA signals a cell-cycle checkpoint response and apoptosis, while interaction of MutS¿ with mismatches signals MMR. We seek to uncover the conformational and dynamic properties of MutS¿-DNA complexes that elicit different cellular fates depending on whether MutS¿ is bound to mismatch or to a DNA lesion. We propose a systematic series of experiments in which we use a combination of bulk and single-molecule fluorescence techniques in conjunction with atomic force microscopy to characterize the binding and dynamic conformational properties of MutS¿- DNA complexes that signal repair versus apoptosis. We will examine the interactions of wildtype and a mutant of MutS¿, which exhibits a separation of function for MMR and DNA damage response,with damaged DNA and with mismatches that exhibit different efficiencies of repair, in the presence and absence of nucleotide cofactors.