Homologous Recombination Outcomes of Double-Strand Break- initiated Events in Saccharomyces cerevisiae
Homologous recombination (HR) is responsible for repairing otherwise lethal DNA double-strand breaks (DSBs) using a homologous donor template. The work in this dissertation, using the budding yeast Saccharomyces cerevisiae as a model system, focuses on the effect of the initiating DSB and sequence homology on the outcomes of repair. First, in Chapter 2 we investigated how the initiating DSB affects HR outcomes. Recombination studies often utilize site-specific enzymes that create a single DSB at a known location. Unlike chemical or environmental DNA damaging agents that cause multiple DSBs genome-wide, these nucleases allow precise analysis of repair intermediates and outcomes of a specific DSB. To date the most common enzymes used are I-SceI and HO which both make DSBs with 3’ overhangs. Although the current HR model is largely based on this one type of DSB, we know DSBs can vary by end structure and initiating cause in vivo. Using our lys2 ectopic assay we explored how the outcomes of recombination vary between a DSB with a 3’ overhang made by I-SceI versus a Zinc Finger Nuclease (ZFN)-generated break with a 5’ overhang. Results of this study indicate the method of DSB induction does not drastically affect HR outcomes. Although these different initiating-breaks produced a slight difference in the distributions of HR products, crossovers (COs) versus noncrossovers (NCOs), there were no detectable differences in the position/length of exchange sequence. Further physical analysis found slight differences in the rate of repair between the different breaks. These findings were significant as they suggest HR is able to handle different types of initiating DSBs and generate similar repair outcomes thus maintaining genome stability. Chapter 3 examines how additional structural challenges between our divergent ectopic substrates affect the outcomes of recombination. By manipulating the donor sequence, we introduced nonhomologous regions between the substrates that generated a gap and/or nonhomologous tails at the DSB. The HR outcomes, including repair frequencies and CO-NCO distributions/frequencies, were observed in the presence and absence of mismatch repair (MMR), as this system is responsible for limiting ectopic recombination (anti-recombination effect). Thus far data suggests these additional structural challenges impede recombination particularly in the presence of MMR activity. The addition of these structures increased the anti-recombination effect of MMR, with gap- and tail- specific effects on HR products. Sequencing of NCO products will be crucial to understanding how the gap and/or tails affect patterns of exchanged sequence, which can give insight into differences in intermediates and mechanisms of recombination. While this study is still ongoing, the findings are significant as they highlight the importance of donor homology and the regulation of ectopic recombination. Overall the work in this dissertation adds to our knowledge of the HR mechanism as defects in this process are linked to human diseases including many types of cancer and genomic disorders.
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