This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.The importance of nucleotide excision repair (NER) is evidenced by the severe human diseases that result from mutations in its component proteins, such as xeroderma pigmentosum (XP). This disorder is characterized by extreme photosensitivity and a greater than 1000-fold increased risk of cutaneous and ocular neoplasms. The incidence of individuals in the United States diagnosed with XP is 1 in 250,000, and the mean onset age of skin cancer is eight years. Our research is focused upon understanding how cells coordinate UV-induced DNA repair and ubiquitin-mediated proteolysis, both crucial for normal functioning of cells exposed to both mutagenic and carcinogenic agents. In Saccharomyces cerevisae, Rad4 is involved in damage recognition during NER and, in conjunction with Rad23, recruits other proteins to the site of damage. Rad23 also acts to stabilize Rad4 by inhibiting multiubiquitin chains from forming, thus preventing its degradation by the 26S proteasome. We identified a synthetic interaction upon UV exposure between Rad4 and Cdc20, a protein that modulates the activity of the anaphase promoting complex (APC/C), a multisubunit E3 ubiquitin ligase complex. The moderately UV sensitive rad4 strain became highly sensitive when cdc20-1 was present.
We aim to determine the mechanistic and physical relationships between Rad4 and Cdc20 with the goal of determining the exact nature of this interaction. Defects in DNA structure will be examined, and a variety of assays designed to detect the ability to overcome DNA damage will be performed. The cell cycle phase interplay of NER and Cdc20 will also be analyzed. Information gathered will contribute to our understanding of how cells coordinate DNA repair and ubiquitin-mediated proteolysis, defects in both implicated in development of malignant cancers.
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