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

染色体DNA中的双链断裂是对所有生物体的持续威胁，如果没有修复或修复， 修复不当的损伤可能导致有害的基因组重排或细胞死亡。细胞对病毒的反应 DNA双链断裂涉及DNA修复因子和信号分子的快速动员 损伤部位，启动DNA修复并触发细胞周期停滞。这些对DNA的反应 中断对于维持基因组的稳定性和丢失这些细胞成分是至关重要的 通路促进了可能导致人类癌症的基因组突变和重排。这个 Mre11/Rad50/Nbs1(Xrs2)(M/R/N(X))复合体在这些事件中起着核心作用，它通过启动DNA双 链断裂修复以及招募和激活信号分子。这项建议解决了 M/R/N(X)复合体的生化活动，总体目标是了解这些活动是如何 与DNA损伤部位的细胞内复合体的功能有关。在之前的工作中，我们使用了重组 人M/R/N复合体在模型DNA底物上的酶活性研究 ATM是DNA损伤信号的主要传导者，它的活动源于DNA双... 链子断了。在目前的提案中，这种生化方法被扩展到也包括S。 Cerevisiae M/R/X和P.Furiosus M/R复合体，以剖析其保守的催化活性 并有效地分离出描述每个组分的关键功能的突变体。利用这一战略 我们将解决发夹结构和共价结构上M/R/N(X)核酸酶活性的底物特异性 蛋白质-DNA体外偶联。我们还将确定RadSO催化结构域的具体作用， 盘绕线圈和锌钩在体外和体内M/R/N(X)-DNA相互作用中的作用。这些实验将 弥合我们对这个复合体的生物化学知识和对生物的观察之间的差距 酵母和哺乳动物细胞中M/R/N(X)突变的后果。 通过描述参与DNA修复和DNA损伤信号的酶的基本机制，我们 可以阐明细胞对DNA损伤的正常反应。这种方法对于理解 癌症进展中涉及自发性或遗传性缺陷的早期事件 并为后续的诊断和治疗试剂提供分子工具。

Double-strand breaks in chromosomal DMAare 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 rearrangementsthat 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 conservedcatalytic 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.