Mrell/Rad50/Nbs1 and DNA double strand break repair

描述(由申请人提供)： 真核细胞暴露在电离辐射下产生DNA光谱 触发信号级联导致DNA修复、细胞周期的损伤 停滞，在某些情况下还会导致细胞凋亡。这些信号通路中的缺陷或 修复机制增加了细胞周期进展的可能性 DNA损伤的存在，这是一种与致癌率增加有关的情况 哺乳动物细胞的转化。本提案针对的是 Mre1-1/Rad50/Nbs1(MJRJN)复合体，它在两个细胞中都起着关键作用 DNA损伤后的周期调节和DNA修复，并在 对DNA双链断裂的反应。从酵母菌实验和 脊椎动物细胞我们知道MJR/N对于非同源末端连接是必不可少的 和姐妹染色单体重组，并在减数分裂重组中发挥作用 以及端粒的维持。此外，Mre11和Nbs1的突变 复合体的成分导致了两种罕见的染色体不稳定 人类的疾病，奈梅亨破裂综合征(NBS)和A-T样障碍， 这会导致极端的辐射敏感性，免疫缺陷和高发病率 恶毒。这些疾病的临床表现与 共济失调-毛细血管扩张症(A-T)，提示MIR/N和A-T突变 蛋白质(ATM)在相同的DNA损伤反应途径中发挥作用。在以前的 我们已经建立了人类M/R/N复合体的表达系统，并 在模型上广泛表征了该络合物的酶活性。 DNA底物。在这里提出的工作中，我们将：a)测试两个模型 MJR/N核酸酶的体内外底物特异性；B)测定 Mre11，Rad50，RAD50突变相关缺陷的生化基础 和Nbs1；以及C)确定Nbs1磷酸化的生化后果 通过ATM，包括对复合体的酶活性和 与其他蛋白质的联系。这些实验将弥合 我们对这个复合体的生物化学知识和对 MJRJN突变在酵母和哺乳动物细胞中的生物学后果。 这项工作将引导我们朝着我们的长期目标--在 ATM和M/R/N在DNA双链断裂修复中的作用机制 以及减数分裂重组和端粒维持。

DESCRIPTION (provided by applicant): Exposure of eukaryotic cells to ionizing radiation generates a spectrum of DNA lesions that trigger signaling cascades leading to DNA repair, cell cycle arrest, and in some cases apoptosis. Defects in these signaling pathways or in the repair machinery increase the probability of cell cycle progression in the presence of DNA damage, a condition linked to increased rates of oncogenic transformation in mammalian cells. This proposal addresses the function of the Mre1 1/Rad50/Nbs1 (MJRJN) complex, which plays a critical role in both cell cycle regulation and DNA repair following DNA damage, and acts specifically in response to DNA double-strand breaks. From experiments in yeast and in vertebrate cells we know that MJR/N is essential for non-homologous end joining and sister chromatid recombination, and also functions in meiotic recombination and telomere maintenance. In addition, mutations in the Mre11 and Nbs1 components of the complex are responsible for two rare chromosomal instability disorders in humans, Nijmegen Breakage Syndrome (NBS) and A-T-like-Disorder, which cause extreme radiation sensitivity, immunodeficiency, and high rates of malignancy. The clinical manifestations of these disorders are very similar to that of Ataxia-Telangiectasia (A-T), suggesting that MIR/N and the A-T-Mutated protein (ATM) function in the same DNA damage response pathway. In previous work we have established an expression system for the human M/R/N complex and have characterized the enzymatic activities of the complex extensively on model DNA substrates. In the work proposed here we will: A) test two models for the substrate specificity of the MJR/N nuclease in vivo and in vitro; B) determine the biochemical basis of defects associated with mutations in Mre1 1, Rad50, and Nbs1; and C) determine the biochemical consequences of Nbs1 phosphorylation by ATM, including effects on the enzymatic activities of the complex and associations with other proteins. These experiments will bridge the gap between our knowledge of the biochemistry of this complex and observations of the biological consequences of MJRJN mutations in yeast and in mammalian cells. This work will lead us toward our long-term goal of understanding at a mechanistic level how ATM and M/R/N function in DNA double-strand break repair as well as in meiotic recombination and telomere maintenance.