Mre11/Rad50/Nbs1 Structural Biology for DNA Damage Responses

 描述(由申请人提供)：Mre11-Rad50-Nbs1(MRN)复合体的缺陷会导致包括癌症在内的人类疾病，以及酵母中严重的DNA损伤表型。MRN与CtIP共同作用，对同源重组修复双链DNA断裂(DSB)以及参与减数分裂、抗体超突变、端粒维持、解救停滞的复制分叉以及通过ATM激酶传递DNA损伤信号是必不可少的。然而，人们对不同MRN功能的机制基础知之甚少。我们提出了三个具体目标来理解与DSB修复和信号转导相关的MRN、CtIP和ATM的结构生化、活性、构象和相互作用。我们将结合先进的生物物理技术，包括原子分辨晶体结构和溶液中的小角X射线散射，与诱变、生物化学和酵母遗传分析相结合。我们的综合方法将检验动态MRN构象和大分子界面控制DSB生物反应的假设。特别是，我们的结果将显著提高对1)mre11DNA结合和核酸酶机制的了解，以及它们对DNA修复途径选择和进展的重要性。2)Rad50如何与DNA结合，并利用其ATPase活性将DNA移交给Mre11，并以变构方式调节Mre11核酸酶活性。3)Rad50患者的突变将促进我们对具有治疗靶向的Rad50蛋白特征的理解。4)CtIP的催化和非催化作用。5) MRN如何在DSB招募和激活自动取款机。我们最新的Mre11抑制剂结果和该领域其他人的工作表明，MRN在DSB修复和信号转导中的作用是开发先进的辅助癌症治疗的可行靶点，这些治疗方法通过与当前的放射和化疗以及其他DNA修复或信号通路中的弱点相结合的合成致死性来发挥作用，这些DNA修复或信号通路是由抑制剂或癌症特异性基因缺陷引起的。因此，我们的综合结果将为理解DNA修复缺陷引起的癌症病因和针对特定MRN活动的先进癌症治疗方法的设计提供一个分子框架。总而言之，项目成果将通过定义对遗传完整性、癌症治疗耐药性和未来癌症治疗至关重要的相互作用、构象和机制，将MRN、CtIP和ATM与细胞结果和人类疾病状态联系起来。

 DESCRIPTION (provided by applicant): Defects in the Mre11-Rad50-Nbs1 (MRN) complex results in human disease including cancer, and severe DNA damage phenotypes in yeast. MRN, acting with CtIP, is essential for the repair of double- stranded DNA breaks (DSBs) by homologous recombinational repair (HRR), as well as acting in meiosis, antibody hypermutation, telomere maintenance, rescue of stalled replication forks, and DNA damage signaling through ATM kinase. Yet, the mechanistic basis for diverse MRN functions is poorly understood. We propose three Specific Aims to understand MRN, CtIP and ATM structural biochemistry, activities, conformations and interactions relevant for DSB repair and signaling. We will couple advanced biophysical technologies, including atomic-resolution crystal structures and small- angle X-ray scattering in solution, with mutagenesis, biochemistry and yeast genetic analyses. Our integrated approaches will test hypotheses that dynamic MRN conformations and macromolecular interfaces control biological responses at DSBs. In particular, our results will significantly advance knowledge of 1) Mre11 DNA-binding and nuclease mechanisms and their importance for DNA repair pathway choice and progression. 2) How Rad50 binds to DNA and uses its ATPase activity to both handoff DNA to Mre11 and allosterically regulate Mre11 nuclease activities. 3) A Rad50 patient mutation that will advance our understanding of therapeutically targetable Rad50 protein features. 4) Catalytic and non-catalytic roles of CtIP. 5) How MRN recruits and activates ATM at DSBs. Our latest Mre11 inhibitor results and work from others in the field suggest that MRN roles in DSB repair and signaling are viable targets for the development of advanced adjunct cancer therapies, which work by synthetic lethality with current radiation and chemotherapies along with weaknesses in other DNA repair or signaling pathways arising from either inhibitors or cancer-specific genetic defects. Thus, our integrated results will provide a molecular framework for understanding cancer etiologies from DNA repair defects and for the design of advanced cancer therapies targeted against specific MRN activities. Collectively, project results will connect MRN, CtIP and ATM to cellular outcomes and human disease-states by defining interactions, conformations and mechanisms critical for genetic integrity, cancer therapy resistance, and future cancer treatments.