Exposure of cells to physical or chemical agents that damage DNA can lead to deleterious consequences, including mutation, cancer or cell death. However, mechanisms are available to properly repair the damage and neutralize the harmful effects of these agents. During the previous funding period, focus was placed on the fission yeast Schizosaccharomyces pombe as a model to understand these mechanisms since this organism shares many cellular and genetic features with mammalian cells. Studies concerned S. pombe rad9 because mutations in this gene cause extreme sensitivity to gamma-rays, UV light, and the DNA replication inhibitor hydroxyurea (HU). Mutant cells also lack the associated checkpoint controls, which can delay entry into mitosis, allowing more time for repair and enhancing resistance to these agents. Using the two-hybrid system, we found that S. pombe rad9p interacts with a c-signal-like protein. We also isolated human and mouse homologues of rad9, and a comparison of the encoded proteins revealed conserved regions which served as targets for in vitro mutagenesis studies. The results indicated that rad9p contains multi-functional and DNA damaging agent response-specific domains, and gamma-ray, UV light and HU resistance could be dissociated. Research emphasis was also placed on the human gene during much of the previous funding period, and it is the focal point of this application. The cDNA and part of the genomic DNA were isolated and characterized, including DNA sequence determination, chromosomal localization, and demonstration of partial complementation of S. pombe rad9::ura4+ defects by the human cDNA. Anti-HRAD9 antibodies were used to show that HRAD9 is a phosphorylated nucleoprotein, extensively modified post-translationally. Co-IP experiments indicated that the human protein interacts physically with BRCA1, HsRAD51, p16 and p53, known tumor suppressor/DNA repair/cell cycle control proteins. The latter results are consistent with HRAD9 being a checkpoint control protein, and suggest testable hypotheses to define its function in humans.
Specific aims are proposed to characterize these physical interactions in more detail, and begin to assess their functional significance. Furthermore, HRAD9 may be regulated by a novel feedback control mechanism, and experiments are proposed to define this system. Taken together, the results should help establish the role of HRAD9 in the cellular response to radiation or chemical exposure, and impact on both basic research and clinical arenas.
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