Stage-Specific Effects of X-Irradiation on Yeast Meiosis

Previous work has shown that cdc13 causes meiotic arrest of Saccharomyces cerevisiae following DNA replication by a RAD9-dependent mechanism. In the present work, we have further investigated the implicit effects of chromosomal lesions on progression through meiosis by exposing yeast cells to X-irradiation at various times during sporulation. We find that exposure of RAD9 cells to X-irradiation early in meiosis prevents sporulation, arresting the cells at a stage prior to premeiotic DNA replication. rad9 meiotic cells are much less responsive to X-irradiation damage, completing sporulation after treatment with doses sufficient to cause arrest of RAD9 strains. These findings thereby reveal a RAD9-dependent checkpoint function in meiosis that is distinct from the G(2) arrest previously shown to result from cdc13 dysfunction. Analysis of the spores that continued to be produced by either RAD9 or rad9 cultures that were X-irradiated in later stages of sporulation revealed most spores to be viable, even after exposure to radiation doses sufficient to kill most vegetative cells. This finding demonstrates that the lesions induced by X-irradiation at later times fail to trigger the checkpoint function revealed by cdc13 arrest and suggests that the lesions may be subject to repair by serving as intermediates in the recombination process. Strains mutant for chromosomal synapsis and recombination, and therefore defective in meiotic disjunction, were tested for evidence that X-ray-induced lesions might alleviate inviability by promoting recombination. Enhancement of spore viability when spo11 (but not hop1) diploids were X-irradiated during meiosis indicates that induced lesions may partially substitute for SPO11-dependent functions that are required for the initiation of recombination.     

Saccharomyces cerevisiae RAD53 (CHK2) but not CHK1 is required for double-strand break-initiated SCE and DNA damage-associated SCE after exposure to X rays and chemical agents

Saccharomyces cerevisiae RAD53 (CHK2) and CHK1 control two parallel branches of the RAD9-mediated pathway for DNA damage-induced G(2) arrest. Previous studies indicate that RAD9 is required for X-ray-associated sister chromatid exchange (SCE), suppresses homology-directed translocations, and is involved in pathways for double-strand break repair (DSB) repair that are different than those controlled by PDS1. We measured DNA damage-associated SCE in strains containing two tandem fragments of his3, his3-Delta5' and his3-Delta3'::HOcs, and rates of spontaneous translocations in diploids containing GAL1::his3-Delta5' and trp1::his3-Delta3'::HOcs. DNA damage-associated SCE was measured after log phase cells were exposed to methyl methanesulfonate (MMS), 4-nitroquinoline 1-oxide (4-NQO), UV, X rays and HO-induced DSBs. We observed that rad53 mutants were defective in MMS-, 4-NQO, X-ray-associated and HO-induced SCE but not in UV-associated SCE. Similar to rad9 pds1 double mutants, rad53 pds1 double mutants exhibited more X-ray sensitivity than the single mutants. rad53 sml1 diploid mutants exhibited a 10-fold higher rate of spontaneous translocations compared to the sml1 diploid mutants. chk1 mutants were not deficient in DNA damage-associated SCE after exposure to DNA damaging agents or after DSBs were generated at trp1::his3-Delta5'his3-Delta3'::HOcs. These data indicate that RAD53, not CHK1, is required for DSB-initiated SCE, and DNA damage-associated SCE after exposure to X-ray-mimetic and UV-mimetic chemicals.     

Inducible error-prone repair in yeast. Suppression by heat shock

The production of reversion mutations in wild-type, diploid Saccharomyces cerevisiae by the alkylating agents N-methyl-N'-nitro- N-nitrosoguanidine (MNNG) and methylnitrosourea (MNU) was suppressed in cells previously treated with a heat shock, or the protein synthesis inhibitor, cycloheximide. The same cells previously treated with a heat shock, or the protein synthesis inhibitor, cycloheximide. The same treatment after mutagen exposure did not lower the induced mutation frequency. In split-dose experiments, a first MNNG exposure prevented subsequent heat (or cycloheximide) treatment from blocking mutation by a second, later mutagen exposure. These data suggest that, in yeast, MNNG or MNU induces an error-prone DNA-repair system, and that this induction is blocked by protein-synthesis inhibitors. The specificity of this system for different types of DNA damage was investigated using a variety of other mutagenic agents. A prior heat shock did not suppress mutation produced by exposure to ethyl methanesulfonate, ethylnitrosourea, 8-methoxypsoralen + UVA, or gamma-radiation. Partial suppression was observed in cells exposed to methyl methanesulfonate or to 254-nm ultraviolet light. These results indicate that, unlike the SOS system of E. coli, this inducible error-prone process of yeast is responsive to only certain mutagens. Heat shock suppression of mutation produced by MNNG exposure was also demonstrated in wild-type haploid cells, as well as haploid strains mutant in representative genes of the RAD52 epistasis group (rad52, rad53, rad54), the RAD3 epistasis group (rad1, rad2, rad3) and the RAD6 epistasis group (rad9, rad18). The rad6 mutant itself was immutable with MNNG and therefore untestable by these techniques. These data indicate that this error-prone repair system is not absolutely dependent on the integrity of the RAD52 (recombination) or the RAD3 (excision) systems, or on at least some parts of the RAD6 system.     

The structure and function of RAD6 and RAD18 DNA repair genes of Saccharomyces cerevisiae

The RAD6 and RAD18 genes of Saccharomyces cerevisiae are required for postreplication repair of discontinuities occurring in newly synthesized DNA following exposure to uv light. In addition, rad6 mutants are highly defective in mutagenesis induced by uv and other DNA damaging agents and in sporulation. RAD6 encodes a protein of 172 amino acids with a highly acidic carboxyl terminus. Deletion of the carboxyl terminal 23 residues, 20 of which are acidic, has little or no effect on uv sensitivity or uv mutagenesis, but sporulation is greatly reduced. Addition of the first four residues of the polyacidic tail restores sporulation to 50% the level observed in RAD+/RAD+ diploids. RAD6 protein has been previously shown to be a ubiquitin-conjugating (E2) enzyme that attaches ubiquitin to histones H2A and H2B in vitro. Our experiments show that deletion of varying lengths of the polyacidic tail of RAD6 protein greatly reduces its ubiquitin-conjugating activity. The RAD18 encoded protein contains features which suggest that it binds DNA and nucleotides. Ten of the 12 cysteine residues occur in regions that could form zinc finger domains for nucleic acid binding. The other interesting feature in RAD18 protein is the presence of a putative nucleotide binding sequence. The possible in vivo functions of the RAD6 and RAD18 proteins are discussed.     

Meiosis Can Induce Recombination in rad52 Mutants of SACCHAROMYCES CEREVISIAE

The RAD52 and RAD50 genes have previously been shown to be required for normal meiotic recombination and for various types of recombination occurring in mitotic cells. Recent evidence suggests that rad52 mutants might be defective in an intermediate recombination step; we therefore examined recombination during meiosis in several rad52 mutants at several different loci and in genetic backgrounds that yield efficient sporulation and synchronous meiosis. Similar to previous reports, spores from rad52 diploids are inviable and meiotic recombination is greatly reduced by rad52 mutations. However, intragenic recombinants were detected when cells were plated on selective media during meiosis; rad52 mutants experience induction of recombination between homologues under these special conditions. The frequencies of recombination at four loci were considerably greater than the mitotic controls; however, they were still at least 20 times lower than corresponding Rad+ strains. The prototrophs induced by meiosis in rad52 mutants were not typical meiotic recombinants because incubation in nutrient-rich medium before plating to selective medium resulted in the complete loss of recombinants. We propose that previously observed single-strand breaks that accumulate in rad52 mutants may be associated with recombinational intermediates that are resolved when cells are returned to selective mitotic media and that the meiosis-induced recombination in rad52 cells does not involve double-strand breaks.     

Repair of UV-irradiated plasmid DNA in Saccharomyces cerevisiae. Inability to complement mutational defects in excision repair by in vitro treatment with Micrococcus luteus UV endonuclease

Excision repair defects of Saccharomyces cerevisiae rad1-1, rad4-4, rad7-1 and rad14 mutants were examined. As previously found, transformation of such cells with UV-irradiated plasmid DNA is poor compared to wild-type yeast. Treatment of UV-irradiated YRp12 plasmid DNA with crude preparations of Micrococcus luteus UV endonuclease before introducing it into rad1-1 cells increased transformation efficiency to wild-type levels. This is consistent with earlier reports of rad1-1 mutants being defective in the incision step of excision repair. However, with purified UV endonuclease little or no rescue occurred when the UV-irradiated plasmid was incised before transformation into rad1-1 or rad4-4 cells. Furthermore, the purified UV endonuclease reduced transformation of rad7-1 and rad14 mutants to levels seen in rad1-1 and rad4-4 cells. In contrast such treatment caused only a small decrease in the transforming ability of UV-irradiated DNA in wild-type cells. These results show that yeast can normally process pre-incised, UV-irradiated DNA and that this activity is absent in rad1-1, rad4-4, rad7-1 and rad14 mutants. Thus, in addition to their previously reported roles in incision, the RAD1, 4, 7 and 14 gene products are also required for repair to continue after the incision of DNA lesions.