UC San Diego

DNA double strand breaks (DSBs) can cause massive genome instability. In eukaryotes, multiple pathways exist to resolve DNA DSBs. One major repair pathway, homologous recombination (HR), utilizes homologous DNA templates to mediate repair. The presence of repetitive elements in the template DNA used for repair can lead to mutagenic repair. Prior studies in Saccharomyces cerevisiae have observed gross chromosomal rearrangements (GCRs) mediated by repetitive DNA from the Ty1 retrotransposon family. Ty1 retrotransposons are highly repetitive sequence elements located throughout the S. cerevisaie genome. In this work, we utilize an endogenous Ty1 element to investigate Ty1- mediated GCRs. We show that the presence of an endogenous high copy repeat sequence in a nonessential chromosome arm increases the rate of GCR formation involving that chromosome arm by nearly 400 fold. Almost all GCRs isolated in this study were mediated by this high copy repeat sequence and ranged from simple monocentric nonreciprocal translocations to nonreciprocal translocations involving template switches to breakage- fusion-bridge (BFB) events. Additionally, despite different mutational defects, the nonreciprocal translocation phenotype appeared to be the predominant GCR phenotype. By adapting a previously developed PCR technique known as multiplex ligation-dependent amplification (MLPA) to study the distribution profiles of GCRs formed in both wild type and mutant strains, we identify ectopic high copy repeat target hotspots. These hotspots mediate the majority of the observed recombinations in wild type strains and may act as fragile sites in the genome. Surprisingly, we find examples of high copy repeat-mediated nonreciprocal translocations in a mutant deficient for RAD52, an important HR gene. Finally, we demonstrate that loss of a histone H3 lysine residue 56 (H3K56) acetylator leads to an increased frequency of aneuploidy that occurs simultaneously with observed GCR events. Taken together, the results indicate Ty1 repeat sequences greatly increase genome instability and that the mechanisms causing increased instability can be further explored using our adapted MLPA technique