Friday, September 13, 2024
9/13/2024
PROJECT SUMMARY/ABSTRACT . DNA replication checkpoint is a cell signaling pathway operating in all eukaryotes that monitors normal S phase progression and in response to perturbed DNA replication, activates cellular responses to prevent irreversible replication fork arrest, genomic instability, and cell death. The checkpoint senses the perturbed replication, maintains the genomic stability under stress and thus functions as an important anticancer barrier. Many anticancer drugs work by interfering with DNA replication and their efficacy is therefore influenced by the checkpoint status of cancer. Despite its importance in disease prevention and cancer chemotherapies, we still do not fully understand the checkpoint initiation process at the replication forks, nor do we know exactly how the checkpoint protects the fork functions under stress. As an established model for studying the cellular mechanisms that are conserved in humans, fission yeast offers several benefits for this research. The goal of this project is to investigate the newly screened checkpoint mutants in fission yeast with particular emphasis on two objectives: (1) understand the mechanistic underpinnings of checkpoint initiation at the perturbed forks, and (2) uncover the essential molecular details of the checkpoint-regulated fork protection. As a starting point, we have developed a combined approach of forward genetics and biochemical analysis and have identified several new mutants with various checkpoint initiation defects under replication stress. We have also screened a large collection of mutants that are defective in fork protection. Guided by our strong preliminary and published data, we will conduct in vivo and in vitro studies under the first objective to investigate how the checkpoint sensor kinase Rad3(ATR) signaling is affected by mutations in the Rad3-Rad26 complex, the RecQ helicase Rqh1, the Smc5/6 complex, and the RPA complex. Under the second objective, we will investigate how the activated checkpoint regulates DNA polymerase e on the leading strand and other yet-to-be identified targets for fork protection. The long-term goal of this research program is to provide a comprehensive understanding of the replication checkpoint that involves three primary areas of inquiry: First, by using our newly improved genetic method, replication proteins with conserved checkpoint functions will be identified. Second, reconstitution of the checkpoint pathway in vitro using purified proteins that can properly recapitulate the in vivo data we and others have obtained. Third, as we show in the studies on Rqh1, conservation of the checkpoint mechanisms in human cells will be evaluated. Overall, this research program will bring much improved clarity to the molecular mechanisms of the replication checkpoint in fission yeast as well as in mammalian cells. The proposed research is significant because of its relevance to genome instability, oncogenesis, and cancer chemotherapies.
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