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

染色体DNA中的双链断裂对所有生物体都是一个持续的威胁， 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双链断裂涉及DNA修复因子以及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 断裂对于维持基因组的稳定性是至关重要的，而这些基因组的细胞成分的丢失是至关重要的。 途径促进基因组突变和复制，这可能导致人类癌症。的 Mre 11/Rad50/Nbs1（Xrs2）（M/R/N（X））复合物通过启动DNA双链反应在这些事件中起着核心作用。 链断裂修复以及募集和激活信号分子。该提案针对 研究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是如何在细胞中发挥作用的。 与细胞中DNA损伤位点的复合物的功能有关。在以前的工作中，我们使用重组 人M/R/N复合物，以阐明复合物对模型DNA底物和对 ATM的活性，DNA损伤信号的主要转换器，起源于DNA双链， 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P. furiosus M/R复合物，以剖析这种保守的催化活性。 酶，并有效地分离突变体，描绘每个组件的关键功能。与这一战略 我们将讨论M/R/N（X）核酸酶活性对发夹结构和共价键的底物特异性， 蛋白质-DNA缀合物体外。我们还将确定RadSO催化结构域的具体作用， 在体外和体内M/R/N（X）-DNA相互作用中的卷曲螺旋和锌钩。这些实验将 在我们对这个复合体的生物化学知识和对生物学的观察之间架起了差距的桥梁。 在酵母和哺乳动物细胞中M/R/N（X）突变的后果。 通过表征参与DNA修复和DNA损伤信号传导的酶的基本机制， 可以阐明正常细胞对DNA损伤的反应。这种方法对于理解 在癌症进展中涉及自发或遗传缺陷的最早事件中， 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.