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

DNA 损伤感知和 ATR、范可尼贫血和 ATM 检查点激活的结构机制

• 批准号: 10639156
• 负责人: NIKOLA P PAVLETICH
• 金额: $ 67.26万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10639156
• 关键词: ATM Signaling Pathway; ATM activation; ATP phosphohydrolase; ATR gene; Acceleration; Address; Amino Acids; Ataxia Telangiectasia; Binding; Biochemical; Cell Cycle Arrest; Cells; Chromatin; Chromosomal Instability; Closure by clamp; Complex; Cryoelectron Microscopy; DNA; DNA Damage; DNA Double Strand Break; DNA Interstrand Crosslinking; DNA Repair; DNA Repair Pathway; DNA Structure; DNA lesion; DNA replication fork; Data; Development; Environment; Ewings sarcoma; FANCD2 protein; Failure; Family; Fanconi Anemia pathway; Fanconi' s Anemia; Genetic; Genome; Goals; Hybrids; Image; Inherited; KRP protein; Lesion; Maintenance; Malignant Neoplasms; Mediating; Metabolism; Methods; Modeling; Molecular Conformation; Monoubiquitination; Mutate; Mutation; Nature; Nijmegen Breakage Syndrome; Oncogenes; Orthologous Gene; Outcome; Pathway interactions; Peptides; Phosphatidylinositols; Phosphorylation; Phosphotransferases; Process; Protein Kinase; Proteins; Publishing; RNA; Role; Signal Transduction; Single-Stranded DNA; Slide; Structure; Syndrome; Therapeutic; Tumor Antigens; Tumor Suppressor Genes; Work; Yeasts; ataxia telangiectasia mutated protein; cancer predisposition; cell growth; crosslink; design; ds-DNA; homologous recombination; novel strategies; nuclease; prevent; programs; protein activation; protein complex; protein purification; protein structure; reconstitution; recruit; repaired; replication stress; response; scaffold; three dimensional structure; translocase; ubiquitin ligase

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.

遗传不稳定性是癌症的一个标志。细胞已经进化出了一套复杂的感知和识别途径。 修复DNA损伤，这是细胞新陈代谢和环境失败的必然结果。 由于压倒性的 DNA 损伤或体细胞或遗传性途径失活，导致修复途径失效 突变，可能导致遗传错误的传播，从而赋予细胞选择优势并驱动 在众多 DNA 修复途径中，对两者的损伤都有反应。 DNA 链尤其重要，因为它们的失败可能导致染色体不稳定，从而导致 加速肿瘤抑制基因的丢失和癌基因的扩增，此类病变包括DNA。 双链断裂 (DSB) 和停滞的复制叉，它们是在复制过程中出现的 DNA 结构 该提案的目的是了解细胞如何感知 DSB 和 停滞的叉子，以及它如何触发具有广泛影响的反应，包括细胞生长停滞和 我们计划使用冷冻电子显微镜（cryo-EM）的方法来确定。 参与这些过程的蛋白质组装体的 3 维结构 - 本质上是详细的图像——将帮助我们更好地理解这些途径是如何工作的，它们是如何在癌症中失败的， 并可能最终帮助确定新的治疗干预方法，这对感知至关重要。 停滞复制叉是 ATR 蛋白激酶，通过磷酸化 ATR 向其他蛋白质发出信号。 及其合作伙伴 ATRIP 感知持久性单链 DNA (ssDNA) 和 dsDNA-ssDNA 连接 – 两个 停滞叉的定义特征 ssDNA 被复制蛋白 RPA 包裹，该蛋白招募 ATR- ATRIP。dsDNA-ssDNA 连接由另一种加载钳子的蛋白质复合物感知，称为 9-1-1， 9-1-1 然后招募 TopBP1 蛋白，该蛋白与 ATR-ATRIP 结合并打开 这是一种由纯化蛋白质重组而成的组装体，我们计划对其进行研究。 我们还计划研究一个相关的组件，其中 TopBP1 被 ETAA1 取代。 我们计划研究的另一个方面是 对失速的货叉进行改造，以促进其传感和修复，并在此过程中提供保护。 功能由遗传性范可尼贫血癌症易感性中突变的 12 种 FANC 蛋白执行 FANCM 重塑叉并招募 9 蛋白复合物（FA 核心复合物）来夹紧。 由 FANCI 和 FANCD2 组成的 DNA，可能会保护分叉 DSB 的感应是通过介导的。 ATM，癌症综合征 DSB 突变的蛋白激酶，与 3- 一起终止。 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。 了解这个过程如何在 3 维结构层面上进行。