THE SACCHAROMYCES-CEREVISIAE RAD9 CHECKPOINT REDUCES THE DNA DAMAGE-ASSOCIATED STIMULATION OF DIRECTED TRANSLOCATIONS

Genetic instability in the Saccharomyces cerevisiae rad9 mutant correl ates with failure to arrest the cell cycle in response to DNA damage. We quantitated the DNA damage-associated stimulation of directed trans locations in RAD9(+) and rad9 mutants, Directed translocations were ge nerated by selecting for His(+) prototrophs that result from homologou s, mitotic recombination between two truncated his3 genes, GAL1::his3- Delta 5' and trp1::his3-Delta 3'::HOcs. Compared to RAD9(+) strains, t he rad9 mutant exhibits a 5-fold higher rate of spontaneous, mitotic r ecombination 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 c hanges, indicating that genomic instability also occurred among non-hi s3 sequences, Both enhanced spontaneous recombination and DNA damage-a ssociated 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 c heckpoint by demonstrating that if rad9 mutants were arrested in G(2) before irradiation, the numbers both of UV and gamma-ray-stimulated re combinants were reduced. The importance of G(2) arrest in DNA damage-i nduced sister chromatid exchange (SCE) was evident by a 10-fold reduct ion 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 R AD9-mediated G(2)-M checkpoint may reduce the frequency of DNA damage- induced translocations is by channeling the repair of double-strand br eaks into SCE.