Cell Cycle and Genetic Requirements of Two Pathways of Nonhomologous End-Joining Repair of Double-Strand Breaks in Saccharomyces cerevisiae

Abstract

In Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break can be repaired by at least two pathways of nonhomologous end joining (NHEJ) that closely resemble events in mammalian cells. In one pathway the chromosome ends are degraded to yield deletions with different sizes whose endpoints have 1 to 6 bp of homology. Alternatively, the 4-bp overhanging 3 ends of HO-cut DNA (5-AACA-3) are not degraded but can be base paired in misalignment to produce ؉CA and ؉ACA insertions. 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 insertions, while almost none had large deletions. When HO expression was confined to the G 1 phase, only 21% were insertions and 38% had large deletions. These results suggest that there are distinct mechanisms of NHEJ 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 which RAD1, RAD2, RAD51, RAD52, RAD54, or RAD57 is deleted; however, deletions of RAD50, XRS2, or MRE11 reduced NHEJ by more than 70-fold when 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 contrast, the rad50 mutation had little effect on the viability of G 1 -induced cells but significantly reduced the frequency of both ؉CA insertions and ؊ACA deletions in favor of larger deletions. Thus, RAD50 (and by extension XRS2 and MRE11) exerts a much more important role in the insertion-producing pathway of NHEJ repair found in S and/or G 2 than in the less frequent deletion events that predominate when HO is expressed only in G 1 .

Cancer predisposition and hematopoietic failure in Rad50S/S mice

“…Genetic analyses in S. cerevisiae establish that the complex's influence can be generally attributed to either structural roles, which primarily influence the DNA recombination functions of the complex, or enzymatic (nucleolytic) roles, which appear to influence DSB end processing or degradation of certain DNA structures. The complex's structural role has been invoked to account for the inability of null Mre11 complex mutants to utilize sister chromatids during recombinational DNA repair, whereas nuclease-deficient mutants do not exhibit this property (Ivanov et al 1992;Moore and Haber 1996;Bressan et al 1999). A molecular basis for such a role is suggested by electron microscopic studies of the human Mre11 complex and structural analysis of the Pyrococcus furiosis Mre11 complex.…”

Random Chromosome Segregation without Meiotic Arrest in Both Male and Female Meiocytes of a dmc1 Mutant of Arabidopsis

“…As for the second hypothesis, if meiotic DSBs are created in the Arabidopsis dmc1 mutant, they are not left unrepaired. Two pathways are known to be involved in DNA DSB repair: one relies on homologous recombination, and the other acts via DNA end joining (Moore and Haber, 1996;Kanaar et al, 1998). DSB repair via homologous recombination with the sister chromatid could be mediated by Arabidopsis Rad51 and associated proteins, in which case the broken chromosome would be healed but no chiasmata would be established.…”

Alternative NHEJ Pathway Components Are Therapeutic Targets in High-Risk Neuroblastoma

“…Despite its nonconservative nature, c-NHEJ is required to suppress translocation formation and generally restores chromosome integrity without rearrangements (16). In addition to c-NHEJ, an alternative NHEJ (alt-NHEJ) mechanism has been described which relies on a distinct set of repair factors (17)(18)(19). PARP1, together with DNA Ligase IIIa (Lig3) or DNA Ligase I (Lig1) binds DSB and initiates end-joining via an alt-NHEJ mechanism that does not utilize c-NHEJ factors (20)(21)(22).…”