Mre11/Rad50/Nbs1 and DNA Double-Strand Break Repair

描述（由申请人提供）：染色体DNA中的双链断裂是所有生物体的持续威胁，未修复或错误修复的损伤可导致有害的基因组重排或细胞死亡。细胞对DNA双链断裂的反应涉及DNA修复因子以及信号分子到损伤位点的快速动员，其启动DNA修复并触发细胞周期停滞。这些对DNA断裂的反应对于维持基因组稳定性至关重要，这些途径的细胞组分的丢失促进了可能导致人类癌症的基因组突变和重排。Mre 11/Rad 50/Nbs 1（Xrs 2）（M/R/N（X））复合物通过启动DNA双链断裂修复以及募集和激活信号分子在这些事件中起着核心作用。该提案涉及M/R/N（X）复合物的生物化学活性，总体目标是了解这些活性如何与细胞中DNA损伤位点处复合物的功能相关。在以前的工作中，我们使用重组人M/R/N复合物来阐明复合物对模型DNA底物的酶活性和对ATM的活性的酶活性，ATM是DNA损伤信号的主要换能器，该信号源自DNA双链断裂。在目前的建议中，这种生物化学方法被扩展到也包括S。酿酒酵母M/R/X和激烈毕赤酵母M/R复合物，以便剖析这种酶的保守催化活性，并有效地分离描述每个组分的关键功能的突变体。通过这种策略，我们将在体外研究M/R/N（X）核酸酶对发夹结构和共价蛋白-DNA结合物的底物特异性。我们还将确定RadSO催化结构域、卷曲螺旋和锌钩在体外和体内M/R/N（X）-DNA相互作用中的具体作用。这些实验将弥合我们对这种复合物的生物化学知识与对酵母和哺乳动物细胞中M/R/N（X）突变的生物学后果的观察之间的差距。通过表征参与DNA修复和DNA损伤信号传导的酶的基本机制，我们可以阐明正常细胞对DNA损伤的反应。这种方法对于理解癌症进展中涉及这些途径中的自发或遗传缺陷的最早事件至关重要，并为后续诊断和治疗试剂提供了分子工具。

DESCRIPTION (provided by applicant): Double-strand breaks in chromosomal DMA are a constant threat to all organisms, and unrepaired or misrepaired lesions can lead to deleterious genomic rearrangements or cell death. The cellular response to DNA double-strand breaks involves a rapid mobilization of DMA repair factors as well as signaling molecules to the damage sites, which initiates DNA repair and triggers cell cycle arrest. These responses to DNA breaks are critical for the maintenance of genomic stability, and loss of the cellular components of these pathways facilitates the genomic mutations and rearrangements that can lead to cancer in humans. The Mre11/Rad50/Nbs1(Xrs2) (M/R/N(X)) complex plays a central role in these events by initiating DNA double strand break repair as well as recruiting and activating signaling molecules. This proposal addresses the biochemical activities of the M/R/N(X) complex with the overall goal of understanding how these activities are related to functions of the complex in cells at sites of DNA damage. In previous work we used recombinant human M/R/N complex to elucidate the enzymatic activities of the complex on model DNA substrates and on the activities of ATM, the primary transducer of the DNA damage signal that originates from DNA double strand breaks. In the current proposal, this biochemical approach is extended to also include the S. cerevisiae M/R/X and P. furiosus M/R complexes in order to dissect the conserved catalytic activities of this enzyme and to efficiently isolate mutants that delineate key functions of each component. With this strategy we will address the substrate specificity of M/R/N(X) nuclease activity on hairpin structures and on covalent protein-DNA conjugates in vitro. We will also determine the specific roles of the RadSO catalytic domain, coiled-coil, and zinc hook in M/R/N(X)-DNA interactions in vitro as well as in vivo. These experiments will bridge the gap between our knowledge of the biochemistry of this complex and observations of the biological consequences of M/R/N(X) mutations in yeast and in mammalian cells. By characterizing the basic mechanisms of enzymes involved in DNA repair and DNA damage signaling, we can elucidate the normal cellular responses to DNA lesions. This approach is essential for an understanding of the earliest events in cancer progression which involve spontaneous or inherited defects in these pathways, and provides the molecular tools for subsequent diagnostic and therapeutic reagents.