2 DIFFERENT TYPES OF DOUBLE-STRAND BREAKS IN SACCHAROMYCES-CEREVISIAEARE REPAIRED BY SIMILAR RAD52-INDEPENDENT, NONHOMOLOGOUS RECOMBINATION EVENTS

In haploid rad52 Saccharomyces cerevisiae strains unable to undergo ho mologous recombination, a chromosomal double-strand break (DSB) can be repaired by imprecise rejoining of the broken chromosome ends. We hav e used two different strategies to generate broken chromosomes: (i) a site-specific DSB generated at the MAT locus by HO endonuclease cuttin g or (ii) a random DSB generated by mechanical rupture during mitotic segregation of a conditionally dicentric chromosome. Broken chromosome s were repaired by deletions that were highly variable in size, all of which removed more sequences than was required either to prevent subs equent HO cleavage or to eliminate a functional centromere, respective ly. The junction of the deletions frequently occurred where complement ary strands from the flanking DNA could anneal to form 1 to 5 bp, alth ough 12% (4 of 34) of the events appear to have occurred by blunt-end ligation. These types of deletions are very similar to the junctions o bserved in the repair of DSBs by mammalian cells (D. B. Roth and J. H Wilson, Mel. Cell. Biol. 6:4293-3304, 1986). When a high level of HO e ndonuclease, expressed in all phases of the cell cycle, was used to cr eate DSBs, we also recovered a large class of very small (2- or 3-bp) insertions in the HO cleavage site. These insertions appear to represe nt still-another mechanism of DSB repair, apparently by annealing and filling in the overhanging 3' ends of the cleavage site. These types o f events have also been well documented for vertebrate cells.