Mre11/Rad50 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菌的严重表型是由Mre 11/Rad 50/Nbs 1（MRN）复合物缺陷引起的。 Mre 11核酸酶与Rad 50 ATP酶的复合物从古生菌到人类都是保守的，并且在S.粟酒属和高等真核生物。 MRN在同源重组修复（HRR）过程中修复DMA双链断裂（DSB）以及通过ATM激酶在减数分裂、抗体超突变、端粒维持和DMA损伤信号传导中起重要作用。 对这些不同的MRN功能的详细机械见解是有限的。 因此，我们提出了两个具体的目标，以进一步了解MRN的结构生物化学，相互作用，和构象变化相关的MRN DNA损伤修复和信号功能。 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，与酵母的遗传和突变分析相结合。 预期的结果将表征功能关键Mre 11和Rad 50蛋白质：蛋白质和蛋白质：DNA界面，构象和相互作用架构。 拟议的耦合生物物理学和遗传学研究将测试有关Mre 11的作用在DNA靶特异性和加工，RadSO的作用在ATP诱导的构象控制和建筑的相互作用，和Nbs 1的C-末端结构域在调制Mre 11和RadSO活动的作用的假设。 此外，在不存在任何MRN复合物成员的情况下观察到的DNA损伤敏感性使得这些结构成为开发抑制剂的基础，以增加细胞对电离辐射和其他DNA损伤剂的敏感性，作为癌症放疗和化疗中的佐剂。 总之，拟议的实验将提供重要的结果，统一理解的分子机制的MNR复合物在遗传完整性和癌症抗性以及MRN结构作为癌症治疗的潜在目标的作用。

DESCRIPTION (provided by applicant): Cancer predispositions in humans and severe phenotypes in yeast result from defects in the Mre11/ Rad50/Nbs1 (MRN) complex. The Mre11 nuclease complex with the Rad50 ATPase is conserved from archaea to humans and regulated by Nbs1 in S. pombe and higher eukaryotes. MRN plays central and essential roles in repairing DMA double-strand breaks (DSBs) during homologous recombination repair (HRR) as well as acting in meiosis, antibody hypermutation, telomere maintenance, and DMA damage signaling through ATM kinase. Detailed mechanistic insights into these diverse MRN functions are limited. We therefore propose two specific aims to further knowledge of MRN structural biochemistry, interactions, and conformational changes relevant to MRN 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 expected results will characterize functionally key Mre11 and Rad50 protein:protein and protein:DNA interfaces, conformations, and interaction architectures. The proposed coupled biophysical and genetic studies will test hypotheses regarding Mre11's role in DNA target specificity and processing, RadSO's role in ATP-induced conformational controls and architectural interactions, and the role of the Nbs1 C-terminal domain in modulating Mre11 and RadSO activities. Furthermore, the DNA damage sensitivity observed in the absence of any MRN complex member makes these structures a basis for the development of inhibitors to increase cellular sensitivity to ionizing radiation and other DNA damaging agents as adjuvants in cancer radiotherapy and chemotherapy. Together, the proposed experiments will therefore provide results important for a unified understanding of the molecular mechanisms underlying the roles of MNR complexes in genetic integrity and cancer resistance as well as MRN structures as potential targets for cancer therapeutics.