Genetic analysis of the DNA damage response

DNA双链断裂（DSBs）是一种潜在的致命染色体病变，如果不进行修复，可能引发基因组不稳定或细胞死亡。真核生物已经进化出了一个复杂的蛋白质网络来感知和修复DSB，这个DSB反应网络的一个关键调节器是ATM（失调性毛细血管扩张突变），这是一种大型的多功能蛋白激酶，负责调节DSB修复，细胞周期检查点反应以及端粒维持。ATM是以一种人类神经退行性疾病命名的，这种疾病与基因组不稳定、免疫缺陷、不育和易患癌症有关。这些不同的活动需要ATM及其重要的辅助因子MRN复合体合作来感知与DNA“末端”相关的染色质结构。它们的相互作用以某种方式激活了ATM/MRN复合物，并将其定位于DNA双链断裂（DSBs）或端粒（染色体的自然末端）的位置。活化的ATM被认为发挥双重作用：既作为在DNA损伤位点组装特定修复复合物的支架，又作为通过磷酸化特定底物调节不同信号通路的激酶。ATM生物学中的一个主要谜团是它如何选择性地对不同类型的染色体“末端”做出反应，区分由DNA损伤引起的端粒和dsb。为了解决这个问题，我们正在研究atm依赖的DNA损伤反应在果蝇，作为一个动物模型。我们发现，在雌性减数分裂过程中，ATM活性的丧失会导致染色体不稳定、端粒缺陷和染色体分离缺陷，从而导致非整倍体配子。这些结果奠定了果蝇作为研究保守ATM功能的良好模型系统。本提案概述了在此基础上建立的策略，应用互补的生化和遗传方法来定义和表征新因素，这些因素将为ATM在DNA损伤反应中的保守作用的分子机制提供新的见解。

DNA double-strand breaks (DSBs) are potentially lethal chromosomal lesions that can trigger genome instability or cell death if they are not repaired. Eukaryotes have evolved a sophisticated network of proteins for sensing and repairing DSBs, A critical regulator of this DSB response network is ATM (ataxia-telangiectasia mutated), a large, multi-functional protein kinase that is responsible for regulating DSB repair, cell cycle checkpoint responses, as well as telomere maintenance. ATM is named for a human neurodegenerative disease associated with genome instability, immunological defects, sterility and predisposition to cancer. These diverse activities require that ATM and its essential co-factor the MRN complex collaborate to sense chromatin structures associated with DNA "ends". Their interactions somehow activate the ATM/MRN complex and localize it to foci at sites of DNA double-strand breaks (DSBs) or telomeres, the natural ends of chromosomes. Activated ATM is thought to play a dual role: both as a scaffold for assembling specific repair complexes at DNA damage sites, and as a kinase that regulates different signaling pathways by phosphorylation of specific substrates.  A major mystery in the biology of ATM is how it selectively responds to different types of chromosome "ends", discriminating between telomeres and DSBs caused by DNA damage. To address this problem, we are studying ATM-dependent DNA damage responses in Drosophila, as an animal model. We showed that loss of ATM activity causes defects that include chromosome instability, telomere defects and chromosome segregation defects during female meiosis resulting in aneuploid gametes. These results establish Drosophila as an excellent model system for studying conserved ATM functions. The present proposal outlines strategies for building on this foundation, applying complementary biochemical and genetic approaches to define and characterize novel factors that will provide new insights into molecular mechanisms underlying conserved roles of ATM in the DNA damage response.