Cellular Responses to Environmentally Driven Replication Stress

About This Grant

PROJECT SUMMARY Numerous environmental agents (hereafter, genotoxins) induce DNA lesions or create other types of barriers that stall replication forks. This can lead to replication stress, and failure to alleviate this stress and restart stalled forks can cause genome instability. Elevated replication stress and the replication-associated mutations that result from genotoxin-induced fork stalling contribute to aging, inflammation, cancer, and numerous other chronic diseases in humans. This project aims to advance understanding of the cellular responses to replication stress. One crucial aspect of the replication stress response involves replication fork reversal, a process that remod- els both the nascent and parental DNA strands to form a four-way junction structure. Fork reversal is carried out by a family of ATP-dependent translocases. Why multiple enzymes with similar activities are involved in this process is not understood. We showed that one of these translocases, HLTF, prevents a remarkably DNA dam- age-tolerant mode of replication by promoting fork reversal and preventing alternative and potentially error-prone modes of DNA synthesis. The unanticipated resilience of the replication fork to various genotoxins in HLTF’s absence may drive mutagenesis and promote the survival of damaged cells. We will investigate the cellular responses to genotoxic and oxidative damage, focusing on the mechanism of replication fork reversal and the impact of loss of fork reversal on genome stability and cell fitness. By combining molecular, biochemical, genomic and proteomic approaches, as well as state-of-the-art single-molecule ap- proaches, we will address the following broad questions: What are the functions of fork reversal and how does it occur in response to environmentally relevant forms of genotoxic damage? What are the specific functions of a central regulator of fork reversal, HLTF, in the face of oxidative and genotoxic DNA damage? Does loss of HLTF and fork reversal increase mutation (rates) in the context of environmental stressors? Our strategy will initially focus in large part on HLTF, elucidating its unique roles. As the project evolves, we will phase in further studies on other remodelers so as to understand, over the long-term, the overall fork reversal process and the unique contributions of each protein to the replication stress response. The knowledge we gain from this research will ultimately facilitate the development of new strategies that i) alleviate the pathological states observed in the absence of the replication stress response, and ii) prevent cancer cells' ability to tolerate DNA damage and develop drug resistance.