Ionizing radiation and restriction enzymes induce microhomology-mediated illegitimate recombination in Saccharomyces cerevisiae

Abstract

DNA double-strand breaks can be repaired by illegitimate recombination without extended sequence homology. A distinct mechanism namely microhomology-mediated recombination occurs between a few basepairs of homology that is associated with deletions. Ionizing radiation and restriction enzymes have been shown to increase the frequency of nonhomologous integration in yeast. However, the mechanism of such enhanced recombination events is not known. Here, we report that both ionizing radiation and restriction enzymes increase the frequency of microhomology-mediated integration. Irradiated yeast cells displayed 77% microhomology-mediated integration, compared to 27% in unirradiated cells. Radiation-induced integration exhibited lack of deletions at genomic insertion sites, implying that such events are likely to occur at undamaged sites. Restriction enzymes also enhanced integration events at random non-restriction sites via microhomology-mediated recombination. Furthermore, generation of a site-specific I-SceI-mediated double-strand break induces microhomology-mediated integration randomly throughout the genome. Taken together, these results suggest that double-strand breaks induce a genome-wide microhomology-mediated illegitimate recombination pathway that facilitates integration probably in trans at non-targeted sites and might be involved in generation of large deletions and other genomic rearrangements.

Rad1, rad10 and rad52 Mutations Reduce the Increase of Microhomology Length during Radiation-Induced Microhomology-Mediated Illegitimate Recombination in Saccharomyces cerevisiae

“…Previously, we found that ionizing radiation-and restriction enzyme-induced NHI events exhibited an increased frequency of microhomology use at the integration junctions (20). We also showed that restriction enzymes enhance the efficiency of NHI at non-restriction sites and that overexpression of ISceI endonuclease induces MHMR events at random genomic locations rather than being targeted to the proximity of the I-SceI-induced DSB.…”

“…Our previous results have shown that radiation protects the singlestranded termini of the integrating DNA from degradation and that the single-stranded termini are required for MHMR (20). We determined whether degradation of the PSS DNA ends may have caused the lower extent of microhomology use in irradiated rad52 mutant cells.…”

“…Both MHMR and the single-strand annealing pathway are accompanied by deletions of the intervening sequences between the homologous sequences, and only one of the repeats remains in the recombined products. However, the minimum homology requirement in single-strand annealing is 29 bp (43) whereas MHMR uses 2-7 bp during nonhomologous integration (20). Several models of MHMR that mimic the mechanism of single-strand annealing have been proposed that involve 5′ to 3′ resection of the DSB and annealing of microhomology sequences, followed by gap-filling and ligation (5,40).…”

REMI in Molecular Fungal Biology

“…This indicates that the cNHEJ pathway is responsible for REMI in this microbe. In S. cerevisiae , REMI results in microhomologymediated recombination (MHMR, Manivasakam and Schiestl 1998 ;Chan et al 2007 ). This suggests the involvement of aNHEJ.…”

“…It is clear that the NHEJ pathway in S. cerevisiae can generate integrations in trans of a cleaved restriction site in the genome. This was concluded from the finding that expression of the endonuclease I-SceI in a S. cerevisiae strain that contained a unique I-SceI recognition site resulted in MHMR throughout its genome (Chan et al 2007 ). Inactivation of ku70 or ku80 has resulted in a relatively higher frequency of homologous recombination in several fungi (Krappmann 2007 ;De Jong et al 2010 ;Salame et al 2012 ;Nakazawa et al 2011 ).…”