There are major gaps in our understanding of mechanisms by which DNA damage is sensed, processed and repaired, and the basis for integration of these events with the cellular life cycle is incompletely understood. Existence of such gaps presents a fundamental problem that precludes our understanding of how cells achieve genome maintenance and prevent cancer in response to environmentally-induced DNA damage. The long-term goal is to elucidate the cellular mechanisms whereby cells coordinate cell cycle progression with sensing, tolerance and repair of DNA damage. The objective in this particular application is to elucidate the roles of an E3 ubiquitin ligase termed 'Rad18'in tolerance of bulky DNA adducts (such as B[a]P- and UV-induced lesions) via Trans-Lesion Synthesis (TLS) and in repair of Double Strand Break (DSB) that arise during S-phase. Based on strong preliminary data, the central hypothesis of this application is that direct phosphorylation of Rad18 by cell cycle and DNA damage-responsive protein kinases is necessary for activating DNA repair, dictating choice of DNA repair pathway and integrating DNA repair with other elements of the cell cycle to enforce genome maintenance. The rationale for the proposed research is that elucidating mechanisms of DNA repair will lead directly to a better understanding of the pathogenesis of cancer.
The specific aims of this proposal are: (1) To determine the mechanisms that initiate Rad18-dependent TLS of bulky DNA adducts. (2) To determine the TLS-independent mechanism(s) of Rad18-mediated DSB repair. (3) To elucidate roles of Rad18 in genome maintenance and tumor suppression in vivo.
For Aim 1, Rad18 phosphorylation and its phosphorylation-dependent regulation of TLS proteins will be studied in vitro and in cultured cells.
Aim 2 will examine the Rad18 E3 ligase-dependence of biochemical signaling pathways involved in DSB sensing and repair.
In Aim 3, mice harboring mutant rad18 alleles will be generated and used to distinguish various effector pathways of Rad18 in genome maintenance and tumorigenesis in vivo. The proposed ideas and research are innovative because they represent a departure from existing paradigms, and seek to provide new paradigms where none exist: Phosphorylation-based regulation of Rad18 is novel and, contrary to existing paradigms, indicates that initiation of Rad18-mediated DNA repair is intimately connected with both checkpoint signaling and cell cycle. The idea that Rad18 has new substrates that are involved in DSB signaling is innovative. The proposed mouse studies are innovative because they will test in vivo roles of Rad18 in genome maintenance for the first time. The proposed work is significant because it will provide new paradigms for Rad18-mediated genome maintenance and tumor suppression that are relevant to human environmental exposures. Moreover, because TLS and DSB repair often confer resistance to chemotherapy and radiotherapy in cancer cells, a deeper understanding of events that activate Rad18-dependent repair could identify druggable targets (e.g. Rad18 or its activating kinases) whose inhibition enhances the efficacy of existing cancer therapies.
The proposed research is relevant to public health because understanding mechanisms of DNA repair will further our understanding of the mechanism and etiology of environmentally-induced cancers, may allow early identification of individuals with cancer propensity, and will likely identify druggable targets whose inhibition will allow better killing of tumor cells by chemotherapies and radiation. The proposed research is relevant to the part of NIH's mission that pertains to developing fundamental knowledge that will help reduce and treat cancer.
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