Abstract
Double strand breaks (DSBs) can occur in the genome from regular cellular processes and environmental, clastogenic agents. Failure and error in repairing DSBs results in genome instability, which can lead to cancer or cell death. Thus, our understanding of DNA repair is intertwined with our understanding of cancer, current therapeutic agents, and gene therapy. There are two major categories of DNA repair: homologous recombination (HR) and non-homologous end joining (NHEJ). The focus of the first half of this thesis is a subtype of homologous recombination repair known as Single Strand Annealing (SSA). SSA is a repair pathway which occurs when a DSB is produced between repeats. In humans, short interspersed repeated sequences (SINES) and long interspersed repeated sequences (LINES), constitute at least 40% of genome (Liddell et al., 2011). To mimic human SSA repair, the Haber laboratory designed yeast (S. cerevisiae) strains that carry two 205-bp URA3 sequences separated by a 2.3kb phage lambda DNA fragment, in which a site-specific DSB can be created. To study mismatch repair (MMR) during SSA, a second yeast strain was created to have 7 mismatches (6 base-pair substitutions and 1-bp insertion/deletion), resulting in a 3% divergence between the repeats.
The product of SSA repair is a single copy of one of the two repeats. Thus, SSA is a highly mutagenic repair pathway. My research confirms that that the helicase Sgs1 decreases SSA by two fold, most likely through a process called heteroduplex rejection (Sugawara et al., 2004). Through sequence analysis of the repaired SSA product, I show that the left fragment is favored during MMR and that heteroduplex rejection and mismatch correction in SSA is mediated by the non-homologous tail.