Ionizing radiation and chemical agents induce strand breaks in DNA, which give rise to mutations and chromosomal alterations that can cause cancer. One of the chief biological defenses against DNA strand breaks is Double-Strand Break Repair (DSBR), a complicated family of biologic pathways that repairs DNA strand breaks. Some modes of DSBR can proceed with full restoration of the DNA sequence and no resulting loss of genetic information, but others result in significant chromosomal loss. The molecular underpinnings of DSBR are being elucidated by researchers in the DNA Repair field, but many questions remain regarding biochemical requirements, timing of the pathways, and even how the most appropriate pathway is selected, given that they are not all equally effective in repairing chromosomes without loss of genetic information. Under this umbrella of interrogation are more specific questions regarding the mechanism for timing and recruitment of proteins that participate in some pathways but not others.
The specific aims of this proposal are to determine in an evolutionarily-conserved eukaryotic model system, the yeast, S. cerevisiae: 1) whether patterns of Rad10 recruitment to Double-Strand Breaks (DSBs) are altered depending on the extent and proximity of DNA sequence homology flanking the DSB site, 2) whether Rad10 recruitment in the "single-strand annealing" (SSA) mode of DSB repair is dependent on the SAW1 gene, and whether Rad10 and Saw1 proteins colocalize at SSA repair sites and 3) whether Rad10 recruitment to DSB sites is dependent on the gene SLX4 and whether Rad10 and Slx4 proteins colocalize at DSB sites.
These aims will be investigated primarily using innovative fluorescence microscopy experiments in which strand breaks will be induced site-specifically in live yeast cells and DNA repair locations will be monitored by fluorescence imaging of the live cells. The behavior of the fluorescently labeled proteins will be compared in strains containing the appropriate combinations of wild-type and mutant genes and varying the DNA sequences flanking the DSBs. Characterization of the functionality of fluorescently-labeled or mutant genes will be investigated with a plasmid-based DNA damage induction/ repair assay which will be analyzed by Southern hybridization. These experiments will address important questions regarding the biochemical requirements bearing on the timing and recruitment of Rad10 in several key modes/contexts of DSBR and delineate the differences in how Rad10 is recruited to DSB sites. The molecular basis for cancer and aging is an important area of research in order to advance clinical strategies to will minimize human suffering. A more detailed understanding of the biochemistry by which cells repair DNA will aid in finding new drug targets for pharmaceuticals that might minimize cancer risk and the ailments associated with aging.
The proposed research will increase our understanding of the mechanism of regulation of a complex biological pathway that protects against cancer and aging known as Double-strand Break Repair. Results of this work may aid in developing cancer prevention strategies and methods to reduce suffering in the elderly by preventing some of the symptoms of aging.
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