The way cells process DNA damage is important since unrepaired or misrepaired lesions can lead to mutation, cancer or cell death. Cells contain multiple pathways that mediate or enhance repair, including excision repair, recombinational repair, mismatch repair or cell cycle progression delay -i.e., checkpoint control. The fission yeast Schizosaccharomyces pombe is used as a model system in this study, because of its similarity to mammalian cells and ease of manipulation, as well as because of the availability of a wide array of mutants that affect radioresistance, DNA repair and cell cycle control. S. pombe rad9 plays a key role in promoting radioresistance since rad9-192 or rad9::ura4 cells are highly sensitive to ionizing radiation and UV light. They also lack G2/M checkpoint control. To define the role of rad9 protein in the context of the whole cell, three extragenic suppressors of the radiosensitivity caused by rad9 mutations were identified by selecting radioresistant derivatives of rad9::ura4 cells. Genetic analyses revealed that each derivative contained a single, unique, non-rad9 gene that mediated the resistance. These extragenic suppressors also partially restored radioresistance to S. Pombe cells containing rad9-192 or rad3-136, the latter being an allele of another checkpoint control gene that bears significant homology to the recently cloned human Ataxia telangiectasia gene, ATM. Also, radioresistance was increased without restoration of checkpoint control. Therefore, the suppressors uncouple radioresistance from cell cycle control governed by rad9 and rad3. The goals of this application focus on defining the role of the suppressors in radioresistance.
Specific aims i nclude suppressor gene cloning, sequence identification and analysis, antibody production for subcellular localization, an analysis of protein-protein interactions, and regulation in relation to protein levels (in combination with studies of mRNA levels) or phosphorylation patterns. Biochemical and genetic studies will be performed to test the potential role of rad9, rad3 or the suppressors in mediating radioresistance through modulation of specific DNA repair pathways. The information gained will help define the role of the three suppressors in radioresistance, and better establish the relationship between cell cycle checkpoint control and other survival promoting pathways.
