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的严重表型是由Mre11/ Rad50/Nbs1 （MRN）复合物缺陷引起的。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，在S. pombe和高等真核生物中受Nbs1调控。MRN在同源重组修复（HRR）中修复DMA双链断裂（dsb）以及减数分裂、抗体高突变、端粒维持和通过ATM激酶的DMA损伤信号传导中发挥核心和重要作用。对这些不同MRN功能的详细机制见解是有限的。因此，我们提出了两个具体的目标，以进一步了解MRN结构生物化学、相互作用以及与MRN DNA损伤修复和信号功能相关的构象变化。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。预期的结果将描述功能关键的Mre11和Rad50蛋白、蛋白质和蛋白质、DNA界面、构象和相互作用结构。拟议的生物物理和遗传学耦合研究将测试有关Mre11在DNA靶标特异性和加工中的作用，RadSO在atp诱导的构象控制和结构相互作用中的作用，以及Nbs1 c端结构域在调节Mre11和RadSO活性中的作用的假设。此外，在没有任何MRN复合体成员的情况下观察到的DNA损伤敏感性使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的佐剂。总之，所提出的实验将为统一理解MNR复合物在遗传完整性和癌症抗性中的作用的分子机制以及作为癌症治疗潜在靶点的MRN结构提供重要的结果。

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.