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.

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