Oxidative DNA damage induces genomic instability and carcinogenesis. DNA double strand breaks (DSBs) are a frequent consequence of oxidative DNA damage. A severe blockade to RNA polymerase II at sites of active transcription, DSBs have profound deleterious and mutational impact on cell survival and genome stability. It remains unclear if and how high fidelity DSB repair is rendered at a transcriptionally active site in nondividing somatic cells that are terminally differentiated, remaining in the quiescent/G0 state. To protect critical regions of the genome, an error-free repair mechanism is expected to protect cells during environmental and intrinsic DNA damage. Otherwise, cumulative DNA damage will be detrimental to normal cellular function and may underlie degenerative neurological diseases and tumorigenesis. Homologous recombination (HR) is an error- free mechanism that repairs DSBs using the paired sister chromatid as a donor of the original genetic information. Mutations of critical genes involved in HR repair lead to breast, ovarian and colon cancers. However, it is unclear if error-free DSB repair can be achieved at transcriptionally active sites in quiescent cells, since there are no replicated and undamaged sister chromatids to act as donors. The goal of this application is to understand the mechanisms governing DSB repair at transcriptionally active sites. Our preliminary studies show that recombination factors such as RAD51, RAD52, and RAD51C are significantly enriched at transcriptionally active damage sites in G0/G1 cells, suggesting a potential involvement of homologous recombinational repair. Assembly and retention of the recombination factors at the damage site requires the Cockayne Syndrome B (CSB) gene. Cockayne Syndrome (CS) patients exhibit progressive neurological degeneration and severe growth failure. We hypothesize that a CSB-dependent mechanism of RNA-templated transcription-associated HR (the CSB-HR pathway) is important to protect coding regions from DSBs. In this project, we will 1) determine how CSB coordinates active chromatin and the HR process to repair damage during transcription; 2) delineate the molecular mechanism of the CSB-HR pathway at the sites of damage in transcribed chromatin; and 3) elucidate the function of the CSB-HR pathway in vivo. Our proposed studies are expected to reveal a novel HR mechanism for somatic cells in the quiescent state. Such a mechanism should explain CS patient clinical features as well as HR deficient tumors associated with DSBs. Moreover, the failure of the CSB-HR mechanism may illustrate how genomic instability causes tumorigenesis initiating from seemingly stable, terminally differentiated cell populations.
Oxidative stress is a key component of the pathogenesis of tumorigenesis. We expect to reveal how mechanisms of defense against oxidative stress contribute to genome stability and prevent DNA damage- induced developmental defects and tumorigenesis. Results from the proposed studies will provide insight into how deficiencies of genome integrity in somatic cells can underlie degenerative neurological diseases as well as tumorigenesis arising from seemingly stable, terminally differentiated (G0) cell populations.
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