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

描述(申请人提供)：染色体DNA的双链断裂是对所有生物体的持续威胁，未修复或错误修复的损伤可能导致有害的基因组重排或细胞死亡。细胞对DNA双链断裂的反应包括快速动员DNA修复因子和信号分子到损伤部位，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的稳定性是至关重要的，而这些途径中细胞成分的丢失有助于基因组突变和重排，从而导致人类癌症。Mre11/Rad50/Nbs1(Xrs2)(M/R/N(X))复合体通过启动DNA双链断裂修复以及募集和激活信号分子在这些事件中发挥核心作用。这项建议涉及M/R/N(X)复合体的生化活动，总体目标是了解这些活动如何与DNA损伤部位细胞中复合体的功能有关。在以前的工作中，我们使用重组人M/R/N复合体来阐明该复合体在模型DNA底物上的酶活性以及ATM的活性，ATM是源于DNA双链断裂的DNA损伤信号的主要转导因子。在目前的建议中，这种生化方法被扩展到也包括酿酒酵母M/R/X和P.Furiosus M/R复合体，以剖析这种酶的保守催化活性，并有效地分离出描述每个组分的关键功能的突变体。通过这一策略，我们将在体外解决发夹结构和共价蛋白质-DNA偶联物上M/R/N(X)核酸酶活性的底物特异性。我们还将在体外和体内确定RadSO催化结构域、卷曲线圈和锌钩在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体的生物化学知识与观察酵母和哺乳动物细胞中M/R/N(X)突变的生物学后果之间的差距。通过表征参与DNA修复和DNA损伤信号转导的酶的基本机制，我们可以阐明正常细胞对DNA损伤的反应。这一方法对于了解癌症进展中涉及自发或遗传缺陷的早期事件至关重要，并为后续的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.