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

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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.