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中严重的DNA损伤表型是由于Mre11-Rad50-Nbs1(MRN)复合体的缺陷造成的。MRN在同源重组修复过程中修复DNA双链断裂，以及通过ATM激酶参与减数分裂、抗体超突变、端粒维持和DNA损伤信号转导等过程中发挥重要作用。然而，对这些不同的MRN功能的详细机械性见解仍然有限。Mre11核酸酶与Rad50 ATPase的复合体在古生物和人类中都是保守的，在S.pombe和人类中受Nbs1调控。我们提出了三个具体的目标来促进对MRN结构生物化学、构象以及与DNA损伤修复和信号功能相关的相互作用的了解。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晶体结构技术，并结合酵母的遗传和突变分析。拟议的生物物理和遗传学综合研究将检验关于Mre11‘S在DNA靶标特异性和加工中的作用，Rad50’S在三磷酸腺苷诱导的构象控制和结构相互作用中的作用，以及NBS1‘S在调节mre11和Rad50活性中的作用的假说。预期的结果将表征关键的Mre11、Rad50和Nbs1蛋白质-蛋白质和蛋白质-DNA界面、构象和相互作用结构。此外，在没有任何一个MRN复合体成员的情况下观察到的DNA损伤敏感性表明，我们的结果将成为一个平台，以测试提高细胞对电离辐射和其他用于癌症放疗和化疗的DNA损伤剂敏感性的抑制剂的效用。总体而言，这些结果将通过定义控制遗传完整性、癌症抗药性、放射抗药性和癌症易感性的相互作用和机制，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.