Failure of DNA damage checkpoint regulation can predispose a cell to become cancerous. The ubiquitin-proteosome system (UPS) is thought to play an important role in regulating responses to DNA damage and DNA repair. In attempting to further define this role, we have observed that a critical component of the regulatory machinery, Rad17, is a UV-induced and ubiquitin-dependent fast-turnover protein. We observed that temporal degradation of Rad17 by Cdh1/APC (ubiquitin-protein ligase) is involved in terminating checkpoint signaling after the completion of the checkpoint response, and that stabilization of Rad17 through functional interference by a non-degradable Rad17 mutant attenuates checkpoint termination and the removal of DNA-damaged cells. We have further observed that impaired proteolysis and persistently elevated Rad17 is a conspicuous feature of malignant melanomas, and that the level of persistence parallels the transition from normal melanocyte through an increasing degree of dysplasia and ultimately to melanoma itself. In addition, stabilized Rad17 impedes genomic integrity, which induces malignant transformation for human primary melanocytes. Our objective in the proposed study is to elucidate details underlying Rad17-mediated checkpoint dysregulation, and examine its impact to the process of melanomagenesis. We seek to assess the extent to which Rad17 persistence may play a pathogenetic role in some cancers. Based on the evidence, we hypothesize that failure of checkpoint regulation is an important facilitator of the development of melanomas and possibly other cancers as well.
Our Specific Aims are the following: (1) To determine the upstream mechanism that regulates Cdh1 and further examine how the interplay between Cdh1/APC and Rad17 terminates checkpoint signaling; (2) to study how Rad17 is regulated by Cdh1/APC during the DNA damage response; and (3) to determine the impact of dysregulated Rad17 proteolysis on carcinogenesis using an established melanoma mouse model and 3D skin reconstruction model. Understanding the biological mechanisms involved in genomic integrity via Rad17 proteolytic regulation and elucidating a potential role for Rad17 proteolysis in melanoma development will be an important antecedent to future studies on the role of impaired DNA damage signaling in oncogenesis, as well as for efforts to identify potential targets for therapeutic intervention.
The proposal is designed to assess the role of Rad17 proteolytic regulation in orchestrating DNA damage checkpoint response and elucidate the mechanism by which Rad17 is ubiquitylated and degraded post after exposure to UV radiation. In addition, this application attempts to determine the pathological impact on how failure on Rad17 proteolysis disrupts the DNA damage checkpoint regulation, which may contribute to the carcinogenesis.
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