Structural Mechanisms of DNA Damage Sensing and Activation of the ATR, Fanconi Anemia, and ATM Checkpoints

基因不稳定是癌症的标志。细胞已经进化出一套复杂的途径， 修复DNA损伤，这是细胞代谢和环境的必然结果。失败 修复途径，由于压倒性的DNA损伤或途径失活的体细胞或遗传 突变，可能导致遗传错误的传播，赋予细胞选择优势， 癌症的发展。在众多的DNA修复途径中，那些对两种损伤都有反应的DNA修复途径， DNA链特别重要，因为它们的失败会导致染色体不稳定， 加速肿瘤抑制基因的丢失和癌基因的扩增。这些病变包括DNA 双链断裂（DSB）和停滞的复制叉，这是在DNA复制过程中产生的DNA结构。 基因组的复制。本提案的目的是了解细胞如何感知DSB， 停滞的叉子，以及它如何引发具有广泛影响的反应，包括细胞生长的停滞， 启动修复程序。我们计划用冷冻电镜的方法来确定 这些过程中所涉及的蛋白质组装体的三维结构。结构信息- 基本上详细的图像-将帮助我们更好地了解这些途径如何工作，它们如何在癌症中失败， 并可能最终帮助确定新的干预治疗方法。感知一种 停滞复制叉是ATR蛋白激酶，通过磷酸化它们向其他蛋白质发出信号。ATR 和它的伙伴ATRIP的正义持久单链DNA（ssDNA）和dsDNA-ssDNA连接-两个 限定了失速叉的特征。ssDNA被复制蛋白RPA包裹，RPA招募ATR- ATRIP。dsDNA-ssDNA连接被另一种蛋白质复合物感知，该蛋白质复合物装载一个称为9-1-1的钳， dsDNA上。9-1-1然后招募TopBP 1蛋白，该蛋白与ATR-ATRIP结合，并打开 磷酸化活性。这是一个由纯化的蛋白质重组而成的组装体，我们计划对其进行研究 冷冻电镜我们还计划研究一个相关的组装，其中TopBP 1被ETAA 1取代 蛋白质，并感觉不同的功能，一个停滞的叉。我们计划调查的另一个方面是 重塑失速的叉子，以促进其传感和修复，以及在此过程中的保护。这些 功能由12种FANC蛋白质执行，这些蛋白质在遗传性范可尼贫血癌症易感性中突变， 综合征FANCM重塑叉和招募9蛋白复合物（FA核心复合物）， 由FANCI和FANCD 2组成的DNA，可能是为了保护叉子。DSB的感知是由 ATM，蛋白激酶突变的癌症综合征共济失调-毛细血管扩张症。DSB结束，连同一个3- 称为MRN的蛋白质复合物激活ATM并启动DSB反应。我们的第三个主要目标是 理解这个过程在三维结构水平上是如何工作的。

Genetic instability is a hallmark of cancer. The cell has evolved an intricate set of pathways that sense and repair DNA damage, which is an inevitable consequence of cellular metabolism and the environment. Failure of a repair pathway, due to either overwhelming DNA damage or pathway inactivation by somatic or inherited mutations, can lead to the propagation of genetic errors that confer a selective advantage to the cell and drive the development of cancer. Among the many DNA repair pathways, those that respond to lesions on both strands of the DNA are particularly important, as their failure can lead to chromosomal instability that can accelerate the loss of tumor suppressor genes and the amplification of oncogenes. Such lesions include DNA double strand breaks (DSBs) and stalled replication forks, which are DNA structures arising during the duplication of the genome. The objective of this proposal is to understand how the cell senses DSBs and stalled forks, and how it triggers a response with wide-ranging effects that include arrest of cell growth and initiation of repair programs. We plan to use the method of cryo-electron microscopy (cryo-EM) to determine the 3-dimensional structures of protein assemblies involved in these processes. Structural information – essentially detailed images – will help us better understand how these pathways work, how they fail in cancer, and may ultimately help identify new approaches to intervene therapeutically. Central to the sensing of a stalled replication fork is the ATR protein kinase that signals to other proteins by phosphorylating them. ATR and its partner ATRIP sense persistent single-stranded DNA (ssDNA) and a dsDNA-ssDNA junction – two defining features of a stalled fork. The ssDNA is coated by the replication protein RPA, which recruits ATR- ATRIP. The dsDNA-ssDNA junction is sensed by another protein complex that loads a clamp, termed 9-1-1, onto dsDNA. 9-1-1 then recruits the TopBP1 protein, which binds to ATR-ATRIP and turns on the phosphorylation activity. This is one assembly, reconstituted from purified proteins, that we plan to investigate with cryo-EM. We also plan to investigate a related assembly, where TopBP1 is replaced with the ETAA1 protein, and which senses different features of a stalled fork. Another aspect we plan to investigate is the remodeling of the stalled fork to facilitate its sensing and repair, and its protection during this process. These functions are carried out by 12 FANC proteins mutated in the inherited Fanconi Anemia Cancer predisposition syndrome. FANCM remodels the fork and recruits a 9-protein complex (FA Core complex) that puts a clamp consisting of FANCI and FANCD2 onto the DNA, likely to protect the fork. The sensing of DSBs is mediated by ATM, protein kinase mutated in the cancer syndrome Ataxia-Telangiectasia. DSB ends, together with a 3- protein complex termed MRN, activate ATM and initiate the DSB response. Our third major goal is to understand how this process works at the level of 3-dimensional structure.