Failure to activate the DNA damage response (DDR) pathway allows cells with unrepaired DNA to divide and can lead to the formation and proliferation of tumors. Impaired DNA repair can increase the incidence of cancer through the formation of deletions, amplifications, and gross chromosomal rearrangements. A hallmark feature of tumor progression is aneuploidy as a result of aberrant chromosome segregation stemming from impaired DNA damage checkpoint activation and/or DNA repair. Importantly, defects in germline DNA repair and DNA damage response also lead to aneuploidy, and as a result, to miscarriages, birth defects, infertility and tumorigenesis. Despite the relevance of both DDR and DNA repair for human health, these mechanisms are not fully understood at the molecular level. Our goal is to elucidate the mechanisms involved in maintaining genomic stability at the molecular level in the nematode C. elegans, an ideal model system for germline studies, amenable to molecular, genetic, biochemical, cytological and computational biology approaches. We have recently identified HIM-20, an uncharacterized D111/Gpatch domain containing protein also present in humans. Partial depletion of HIM-20 results in sensitivity to ionizing radiation and a delay in the repair of meiotic double strand breaks. HIM-20 colocalizes and interacts with crossover promoting proteins. We propose that HIM-20 is involved in DNA repair and acts to promote crossover formation. We will determine the mechanism of function of HIM-20 in DNA repair by examining both the progression and output of recombination in him-20 mutants; the response of him-20 mutants to DNA damage; the interdependencies driving the localization pattern of HIM-20; identifying its binding partners; and determining its DNA substrate binding specificities in vitro. These studies will provide critical insight into the procss by which HIM-20 and its human ortholog promote genome stability. Through combined genetic, molecular, cytological and biochemical approaches, we will determine the mechanism of function for ZTF-8, a novel and conserved protein that our studies have shown interacts with a component of the 9-1-1 DDR complex and is required for DDR and DSB repair. These studies will shed new light on our understanding of the DDR and DSB repair pathways in the germline. Finally, we will identify the genetic interaction network required for proper chromosome segregation and apoptosis in the nematode C. elegans. We will test pair-wise combinations of genetic mutations in DNA repair and DDR genes with depletion of germline-enriched genes by RNAi in a high-throughput format designed and proven to detect chromosome missegregation and cells undergoing apoptosis. Hierarchical clustering of genetic interaction profiles associated with each query will order genes to identify those that are functionally related, predict biochemical pathways and protein complexes, assign function to uncharacterized genes, and reveal the large-scale structure of the biological network driving genome stability. Taken together, this application will provide significant new insights into the molecular mechanisms regulating genome stability.
! Defects in germline DNA repair and DNA damage response lead to miscarriages, birth defects, infertility and tumor formation. This application takes advantage of the ease of genetic, cytological, molecular, biochemical and computational biology analysis in the worm C. elegans to investigate the molecular mechanisms of function for DNA repair and DNA damage response proteins also present in humans. These studies will significantly contribute to our understanding of how these critical processes regulate human reproductive health and provide potential new targets for diagnosing cancer susceptibility and cancer drug therapy.
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