The Saccharomyces cerevisiae RAD9 Checkpoint Reduces the DNA Damage-Associated Stimulation of Directed Translocations

Abstract

Genetic instability in the Saccharomyces cerevisiae rad9 mutant correlates with failure to arrest the cell cycle in response to DNA damage. We quantitated the DNA damage-associated stimulation of directed translocations in RAD9؉ and rad9 mutants. Directed translocations were generated by selecting for His ؉ prototrophs that result from homologous, mitotic recombination between two truncated his3 genes, GAL1::his3-⌬5 and trp1::his3-⌬3::HOcs. Compared to RAD9 ؉ strains, the rad9 mutant exhibits a 5-fold higher rate of spontaneous, mitotic recombination and a greater than 10-fold increase in the number of UV-and X-ray-stimulated His ؉ recombinants that contain translocations. The higher level of recombination in rad9 mutants correlated with the appearance of nonreciprocal translocations and additional karyotypic changes, indicating that genomic instability also occurred among non-his3 sequences. Both enhanced spontaneous recombination and DNA damage-associated recombination are dependent on RAD1, a gene involved in DNA excision repair. The hyperrecombinational phenotype of the rad9 mutant was correlated with a deficiency in cell cycle arrest at the G 2 -M checkpoint by demonstrating that if rad9 mutants were arrested in G 2 before irradiation, the numbers both of UV-and ␥-ray-stimulated recombinants were reduced. The importance of G 2 arrest in DNA damageinduced sister chromatid exchange (SCE) was evident by a 10-fold reduction in HO endonuclease-induced SCE and no detectable X-ray stimulation of SCE in a rad9 mutant. We suggest that one mechanism by which the RAD9-mediated G 2 -M checkpoint may reduce the frequency of DNA damage-induced translocations is by channeling the repair of double-strand breaks into SCE.It has been postulated that DNA damage-induced cell cycle arrest at cell cycle checkpoints maintains genomic stability by allowing time for DNA repair prior to the replication or division of damaged chromatids (67, 68). Consistent with this idea, mutations in genes controlling cell cycle arrest at the G 1 -S checkpoint and G 2 -M checkpoint confer enhanced genetic instability. For example, p53 mutations, which confer deficiencies in the G 1 -S checkpoint, are correlated with enhanced spontaneous and UV-stimulated amplification of CAD genes (35, 73). Cells cultured from patients with ataxia telangiectasia that are deficient in cell cycle arrest at both the G 1 -S and G 2 -M cell cycle checkpoints (7, 49) also exhibit higher frequencies of chromosomal rearrangements, including translocations (37) and deletions (38), and chromosome end-to-end joining (41).In Saccharomyces cerevisiae, DNA-damaging agents stimulate mitotic, homologous recombination and induce cell cycle arrest at cell cycle checkpoints (31, 59). For example, DNA damage-associated recombination between his3 fragments positioned at predetermined loci can result in chromosomal rearrangements, including translocations (18, 19), duplications (16), and deletions (54). HO endonuclease-generated doublestrand breaks (DSBs) stimulate ectopic gene conversi...

A genome-wide screen for methyl methanesulfonate-sensitive mutants reveals genes required for S phase progression in the presence of DNA damage

“…We combined our data for MMS and HU sensitivity with the reported data for UV and IR sensitivity to cluster the MMS-sensitive mutants on the basis of their cross-sensitivity to UV, IR, and HU (Table 2). Because both these genome-wide studies missed known UV-or IR-sensitive mutants, we supplemented the data with literature reports (4,5,49,(51)(52)(53)(54)(55)(56)(57)(58)(59)(60)(61)(62). We noted several interesting properties of these mutants.…”

Nuclear oscillations and nuclear filament formation accompany single-strand annealing repair of a dicentric chromosome in Saccharomyces cerevisiae

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The Dot1 Histone Methyltransferase and the Rad9 Checkpoint Adaptor Contribute to Cohesin-Dependent Double-Strand Break Repair by Sister Chromatid Recombination in Saccharomyces cerevisiae

“…The checkpoint adaptor Rad9 functions in SCR: Using genetic assays based on his3 truncated genes it has been proposed that Rad9 promotes DSB repair by SCR (Fasullo et al 1998) and genetic studies of DNA damage sensitivity have placed DOT1 in the RAD9 epistasis group (Wysocki et al 2005;Conde and SanSegundo 2008). In addition, it has been reported that Dot1-mediated histone H3K79 methylation is required for the recruitment of the Rad9 checkpoint adaptor protein to an irreparable HO-induced DSB at the chromosomal MAT locus (Wysocki et al 2005).…”