Mre11/Rad50/Nbs1 Structural Biology for DNA Damage Responses

描述（由申请人提供）：人类的癌症易感性和酵母的严重DNA损伤表型是由Mre11-Rad50-Nbs1 （MRN）复合物的缺陷引起的。MRN在同源重组修复过程中修复DNA双链断裂（DSBs），并在减数分裂、抗体超突变、端粒维持和通过ATM激酶的DNA损伤信号传导中发挥核心和重要作用。然而，对这些不同MRN功能的详细机制了解仍然有限。含有Rad50 atp酶的Mre11核酸酶复合体从古细菌到人类都是保守的，并且在S. pombe和人类中受Nbs1的调控。我们提出了三个特定的目标，以推进与DNA损伤修复和信号功能相关的MRN结构生物化学，构象和相互作用的知识。为了实现这些目标，我们将应用先进的生物物理技术，包括同步加速器溶液x射线散射和原子分辨率晶体结构技术，以及酵母的遗传和突变分析。拟议的综合生物物理和遗传学研究将测试有关Mre11在DNA靶标特异性和加工中的作用，Rad50在atp诱导的构象控制和结构相互作用中的作用，以及Nbs1在调节Mre11和Rad50活性中的作用的假设。预期结果将表征功能关键的Mre11， Rad50和Nbs1蛋白-蛋白质和蛋白质- dna界面，构象和相互作用结构。此外，在MRN复合物中没有任何一个成员的情况下观察到的DNA损伤敏感性表明，我们的研究结果将形成一个平台，以测试用于癌症放疗和化疗的抑制剂的效用，这些抑制剂可以增加细胞对电离辐射和其他DNA损伤剂的敏感性。总的来说，研究结果将通过定义相互作用和控制遗传完整性、癌症抗性、放疗抗性和癌症易感性的机制，将MRN与细胞结果和人类疾病联系起来。

DESCRIPTION (provided by applicant): Cancer predispositions in humans and severe DNA damage phenotypes in yeast result from defects in the Mre11-Rad50-Nbs1 (MRN) complex. MRN plays central and essential roles in repairing DNA double-strand breaks (DSBs) during homologous recombination repair as well as acting in meiosis, antibody hypermutation, telomere maintenance, and DNA damage signaling through ATM kinase. Yet, detailed mechanistic insights into these diverse MRN functions remain limited. The Mre11 nuclease complex with the Rad50 ATPase is conserved from archaea to humans and is regulated by Nbs1 in S. pombe and humans. We propose three Specific Aims to advance knowledge of MRN structural biochemistry, conformations, and interactions relevant to DNA damage repair and signaling functions. To accomplish these Aims, we will apply advanced biophysical techniques, including synchrotron solution X-ray scattering and atomic resolution crystal structure technologies in concert with genetic and mutational analyses in yeast. The proposed integrated biophysical and genetic studies will test hypotheses regarding Mre11's role in DNA target specificity and processing, Rad50's role in ATP-induced conformational controls and architectural interactions, and Nbs1's role in modulating Mre11 and Rad50 activities. The expected results will characterize functionally key Mre11, Rad50 and Nbs1 protein-protein and protein-DNA interfaces, conformations, and interaction architectures. Furthermore, the DNA damage sensitivity observed in the absence of any one member of the MRN complex suggests that our results will form a platform to test the utility of inhibitors that increase cellular sensitivity to ionizing radiation and other DNA damaging agents used for cancer radiotherapy and chemotherapy. Overall the results will connect MRN to cellular outcomes and human disease by defining interactions and mechanisms controlling genetic integrity, cancer resistance, radiotherapy resistance, and predispositions to cancer. PUBLIC HEALTH RELEVANCE: Major goals for cancer research for the past decade have been the identification of the molecular underpinnings of cancer and the development of novel approaches to intervention. A major advance has been the characterization of the Mre11-Rad50-Nbs1 (MRN) complex as a critical suppressor of tumorigenesis and a resistance factor for current therapies. The proposed research will provide insights into the molecular mechanisms for MRN functions relevant to both cancer avoidance and interventions.