DNA double strand breaks can occur from various sources and the timely and accurate repair of these breaks is critical to maintain the genomic integrity of the cell. Break-induced replication (BIR) is a repair pathway that has been shown to repair DSBs where only one end of the break can locate homology, similar to ends seen at collapsed replication forks or eroded telomeres. BIR progresses by an unusual bubble-like intermediate. The asynchrony between the synthesis of leading and lagging strand synthesis during BIR leads to the accumulation of long single-stranded DNA (ssDNA) behind the bubble. This mechanism leads to the conservative inheritance of newly synthesized DNA. BIR repair can lead to increased mutations, loss of heterozygosity and gross chromosomal rearrangements. In this thesis I investigated the deleterious effects of the ssDNA formed during BIR. Using yeast, Saccharomyces cerevisiae, I showed that the regulation of Rad51 that binds ssDNA during BIR is important to prevent the accumulation of toxic joint intermediates. Here, I demonstrate that a known Rad51-interacting protein, Srs2, plays two key roles in counteracting the accumulation of lethal recombination intermediates. First, Srs2 dislodges Rad51 from long ssDNA formed during DSB repair and therefore prevents promiscuous strand invasions that generate lethal joint molecules. Second, Srs2 helicase dismantles toxic intermediates that have already formed. We also demonstrate that the structure-specific endonucleases, Mus81 and Yen1, can resolve toxic joint molecules formed in the absence of Srs2, thus promoting cell survival.
The other goal of this thesis was to study the effects of ssDNA accumulated during BIR in the formation of base-substitution mutagenesis. I test whether this ssDNA is mutagenic by analyzing BIR with and without the presence of DNA damaging agents, including methyl methanesulfonate (MMS) and APOBEC3A. I observed a hypermutagenic effect of BIR with respect to base- substitutions in both cases. Importantly, BIR synergizes with ssDNA damaging agents to produce mutation clusters similar to those previously observed in cancer. I also report the critical role translesion polymerase Polζ plays in the formation of base-substitutions resulting from BIR. Finally, I have discovered a completely novel, UNG1-dependent mechanism of supposed error-free bypasses of APOBEC-induced DNA lesions during BIR that promotes chromosomal rearrangements.