Knowledge Bank

During replication DNA can sustain spontaneous double strand breaks (DSBs). The major pathway of DSB repair is genetic recombination which can produce both error proof and error prone repair depending on what sub-pathway is chosen. Single strand annealing (SSA) is an error prone repair mechanism for fixing breaks that occur at non-tandem direct repeats (repeats with a unique sequence in between). This mechanism causes deletions of the region between the repeats. Such deletions have been seen in cancer cells, but their mechanism has not been defined. It has been previously observed that the yeast histone remodeler Hip1 (Human HIRA) plays a role in biasing DSB repair towards chromosomal deletion through SSA. However, there has been no defined epistatic pathway for how Hip1 influences deletions through SSA. Hip1 is a replication independent histone chaperone that functions to deposit histones after DNA damage repair. My project focused on defining the epistatic pathway for hip1+ with the aim of identifying the role of hip1+ in SSA. Using the power of yeast genetics double and triple mutant strains were created by crossing a hip1 deletion (hip1∆) strain with a variety of mutant recombination and replication genes. These mutants were analyzed for their sensitivity to various DNA damage drugs known to affect specific repair pathways. Fluorescence imaging was also used to visualize DNA damage. In both cases the results were compared to single mutant and wild-type outcomes using the same experimental measures. This work identified a unique interaction between hip1 and two key components in DNA recombination and repair: rad51 and rad52. Rad51 and Rad52 are key regulators of homologous recombination, the major pathway of DNA damage repair, and mutants are highly sensitive to DNA damaging drugs. Remarkably, deletion of hip1 suppresses this sensitivity phenotype of both rad51 and rad52. We speculate that hip1 may be acting as a transcriptional repressor for additional repair pathways that can lead to chromosomal repair in the absence of rad51 and rad52. Hip1 is known to interact with various transcriptional repressors. Due to the high conservation of function between yeast and human repair pathways these findings are translatable to human disease.