CELL-CYCLE AND GENETIC REQUIREMENTS OF 2 PATHWAYS OF NONHOMOLOGOUS END-JOINING REPAIR OF DOUBLE-STRAND BREAKS IN SACCHAROMYCES-CEREVISIAE

In Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break can be repaired by at least two pathways of nonhomologous end jo ining (NHEJ) that closely resemble events in mammalian cells. In one p athway the chromosome ends are degraded to yield deletions with differ ent sizes whose endpoints have 1 to 6 bp of homology, Alternatively, t he 4-bp overhanging 3' ends of HO-cut DNA (5'-AACA-3') are not degrade d but can be base paired in misalignment to produce +CA and +ACA inser tions, When HO was expressed throughout the cell cycle, the efficiency of NHEJ repair was 30 times higher than when HO was expressed only in G(1). The types of repair events were also very different when HO was expressed throughout the cell cycle; 78% of survivors had small inser tions, while almost none had large deletions, When HO expression was c onfined to the G, phase, only 21% were insertions and 38% had large de letions, These results suggest that there are distinct mechanisms of N HEJ repair producing either insertions or deletions and that these two pathways are differently affected by the time in the cell cycle when HO is expressed, The frequency of NHEJ is unaltered in strains from wh ich R LDI, RAD2, RAD51, RAD52, RAD54, or RAD57 is deleted; however, de letions of RAD50, XRS2, or MRE11 reduced NHEJ by more than 70-fold whe n HO was not cell cycle regulated, Moreover, mutations in these three genes markedly reduced +CA insertions, while significantly increasing the proportion of both small (-ACA) and larger deletion events, In con trast, the rad50 mutation had little effect on the viability of G(1)-i nduced cells but significantly reduced the frequency of both +CA inser tions and -ACA deletions in favor of larger deletions, Thus, RAD50 (an d by extension XRS2 and MRE11) exerts a much more important role in th e insertion-producing pathway of NHEJ repair found in S and/or G(2) th an in the less frequent deletion events that predominate when HO is ex pressed only in G(1).