Mechanistic studies of DNA repair and damage response

DNA修复和损伤反应的机制研究

• 批准号: 7924093
• 负责人: DOROTHY A ERIE
• 金额: $ 23.23万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7924093
• 关键词: ATP phosphohydrolase; Affinity; Alanine; Alkylating Agents; Apoptosis; Atomic Force Microscopy; Binding; Biochemical; Cell Cycle Checkpoint; Cell Death; Cell Survival; Cisplatin; Complement; Complex; DNA; DNA Binding; DNA Binding Domain; DNA Damage; DNA Repair; DNA biosynthesis; DNA lesion; DNA-Protein Interaction; Eukaryota; Excision; Exhibits; Fluorescence; Fluorescence Anisotropy; Frequencies; Genes; Genetic Recombination; Genome; Glutamates; Goals; Grant; Hereditary Nonpolyposis Colorectal Neoplasms; Homologous Gene; Human; Hydrolysis; Individual; Label; Link; Maintenance; Malignant Neoplasms; Methods; Mismatch Repair; Mismatch Repair Gene Inactivation; Molecular Conformation; Monitor; Mutation; Nucleotides; Pathway interactions; Phenotype; Prokaryotic Cells; Property; Protein Dynamics; Proteins; Regulation; Resistance; Series; Signal Transduction; Techniques; Vertebral column; Yeasts; chemotherapy; cofactor; cytotoxic; dimer; in vivo; mutant; protein protein interaction; repaired; research study; response; single molecule; single-molecule FRET

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.

这项建议的总体目标是阐明MutS？的结构和动力学性质。 启动DNA错配修复(MMR)与启动DNA的DNA复合体 损伤诱导的细胞凋亡。MMR是纠正DNA合成错误的机制 复制后，它是维持基因组完整性的核心。MMR 蛋白质还参与了其他几种DNA交易，包括DNA损伤感知。在……里面 除了显著增加突变频率外，MMR基因的失活 减少细胞凋亡，增加细胞存活率，并导致对化疗的抗药性。在人类身上， 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同源基因结合到一个 不匹配。随后，MutL同系物与MutS同系物在依赖于ATP的情况下相互作用 如何和协调蛋白质-蛋白质相互作用，以信号切除和重新合成 新合成的含有错误核苷酸的DNA链。引发DNA损伤- 真核生物中诱导的细胞凋亡遵循非常相似的启动途径。MutS同源 MSH2-MSH6(MutS？)优先与DNA损伤结合，随后与 MutL同源物MLH1-PMS2(酵母中的MLH1-PMS1；MutL？)，但不是启动DNA 错配修复，这些相互作用激活细胞周期检查点，并发出凋亡信号。这个 这项资助的中心目标是了解MutS与受损DNA信号的相互作用是如何 细胞周期检查点反应与细胞凋亡，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 根据MutS是否必然错配而导致不同细胞命运的复合体 或者是DNA损伤。我们提出了一系列系统的实验，在这些实验中，我们使用 结合原子的体相和单分子荧光技术 力显微镜表征MutS？-的结合和动态构象性质 发出修复信号的DNA复合体而不是细胞凋亡。我们将研究野生型的相互作用 和MutS的一个突变体，它表现出对MMR和DNA损伤的功能分离 对DNA受损和错配表现出不同修复效率的反应， 核苷酸辅因子的存在和缺失。