Mechanistic Characterization of the Replication Stress Response

项目总结

PROJECT SUMMARY Accurate DNA replication is a fundamental process that governs the survival of every organism. Cellular DNA is under constant assault from various sources. The cellular replication machinery frequently encounters and is severely vulnerable to agents that stall its advancement, leading to replicative errors, development of mutations, and various forms of genetic instability. As a result of this, elevated levels of replication stress is a characteristic hallmark of various disease conditions. It is therefore critical to understand in molecular detail the varied mechanisms by which cells respond to and adequately repair damaged DNA during replication stress. Although we know a lot about how breaks in DNA are repaired, there is a major gap in our understanding of how cells adequately respond to replication stress in part due to the lack of genetic and biochemical tools to probe these processes. To gain further insights into the processes involved in the replication stress response, and in order to identify and characterize the panoply of genes required for this pathway, during my postdoc I performed whole genome screens in multiple cell lines following perturbations with low doses of replication stress-inducing agents. From these screens I generated a novel dataset that includes multiple genes that have yet to be linked with genome instability. Several newly identified genes were linked to chromatin responses, replication fork maintenance pathways, regulation of nucleotide biosynthesis and others. Of note, I identified the Protexin complex, consisting of the single stranded DNA binding protein SCAI and the DNA polymerase REV3. Protexin was critical for maintaining genomic instability by regulating single stranded DNA accumulation through unknown mechanisms. These screens also revealed a striking role for RNA dependent processes in the replication stress response, a novel layer of regulation that had not been appreciated before now. Among our top hits, we identified several novel RNA helicases and RNA-binding factors, as well as several non-coding RNA molecules, demonstrating a crucial, intimate link between RNA-dependent processes and adequate maintenance of genome stability. My lab will take advantage of this vast resource of newly identified factors to characterize novel genome maintenance mechanisms. Investigating these novel factors will allow us to decipher in detail the concerted, multi-layered repair response and fork restoration control upon exposure to DNA damage. We will (1) characterize the mechanism of single stranded accumulation following replication stress. 2) Identify mechanisms by which RNA-modifying enzymes function in the replication stress response, and 3) elucidate roles for non-coding RNA genes during the replication stress Response. Completion of these research projects will grant us significant and fundamental novel insights into how cellular genomes are maintained in the face of damaging insults, grant us improved mechanistic understanding of the principles of cancer pathogenesis in greater detail and open up novel avenues for exploiting defective DNA repair processes in the treatment of cancers.