Saccharomyces cerevisiae rad51 mutants are defective in DNA damage-associated sister chromatid exchanges but exhibit increased rates of homology-directed translocations.

Saccharomyces cerevisiae Rad51 is structurally similar to Escherichia coli RecA. We investigated the role of S. cerevisiae RAD51 in DNA damage-associated unequal sister chromatid exchanges (SCEs), translocations, and inversions. The frequency of these rearrangements was measured by monitoring mitotic recombination between two his3 fragments, his3-Delta5' and his3-Delta3'::HOcs, when positioned on different chromosomes or in tandem and oriented in direct or inverted orientation. Recombination was measured after cells were exposed to chemical agents and radiation and after HO endonuclease digestion at his3-Delta3'::HOcs. Wild-type and rad51 mutant strains showed no difference in the rate of spontaneous SCEs; however, the rate of spontaneous inversions was decreased threefold in the rad51 mutant. The rad51 null mutant was defective in DNA damage-associated SCE when cells were exposed to either radiation or chemical DNA-damaging agents or when HO endonuclease-induced double-strand breaks (DSBs) were directly targeted at his3-Delta3'::HOcs. The defect in DNA damage-associated SCEs in rad51 mutants correlated with an eightfold higher spontaneous level of directed translocations in diploid strains and with a higher level of radiation-associated translocations. We suggest that S. cerevisiae RAD51 facilitates genomic stability by reducing nonreciprocal translocations generated by RAD51-independent break-induced replication (BIR) mechanisms.

Radiosensitive and mitotic recombination phenotypes of the Saccharomyces cerevisiae dun1 mutant defective in DNA damage-inducible gene expression.

The biological significance of DNA damage-induced gene expression in conferring resistance to DNA-damaging agents is unclear. We investigated the role of DUN1-mediated, DNA damage-inducible gene expression in conferring radiation resistance in Saccharomyces cerevisiae. The DUN1 gene was assigned to the RAD3 epistasis group by quantitating the radiation sensitivities of dun1, rad52, rad1, rad9, rad18 single and double mutants, and of the dun1 rad9 rad52 triple mutant. The dun1 and rad52 single mutants were similar in terms of UV sensitivities; however, the dun1 rad52 double mutant exhibited a synergistic decrease in UV resistance. Both spontaneous intrachromosomal and heteroallelic gene conversion events between two ade2 alleles were enhanced in dun1 mutants, compared to DUN1 strains, and elevated recombination was dependent on RAD52 but not RAD1 gene function. Spontaneous sister chromatid exchange (SCE), as monitored between truncated his3 fragments, was not enhanced in dun1 mutants, but UV-induced SCE and heteroallelic recombination were enhanced. Ionizing radiation and methyl methanesulfonate (MMS)-induced DNA damage did not exhibit greater recombinogenicity in the dun1 mutant compared to the DUN1 strain. We suggest that one function of DUN1-mediated DNA damage-induced gene expression is to channel the repair of UV damage into a nonrecombinogenic repair pathway.

Expression of Saccharomyces cerevisiae MATa and MAT alpha enhances the HO endonuclease-stimulation of chromosomal rearrangements directed by his3 recombinational substrates.

Radiation resistance in Saccharomyces cerevisiae is greater in a/alpha diploids than in aa or alpha alpha diploids, and higher levels of radiation resistance correlates with more mitotic recombination. Specifically, we investigated whether the stimulation of directed translocations, inversions, and unequal sister chromatid exchanges (SCEs) by HO endonuclease-induced double-strand breaks (DSBs) is enhanced in a/alpha cells. These rearrangements result from mitotic recombination between two truncated his3 genes, his3-delta 5' and his3-delta 3'::HOcs, positioned on non-homologous chromosomes or positioned in juxtaposition on the same chromosome in inverted or direct orientation. Mitotic recombination was initiated by HO endonuclease-induced DSBs at the HO cut site (HOcs) located at his3-delta 3'::HOcs, and His+ recombinants were selected. In MATa-inc haploid strains, which do not switch mating-type, the DSB reduced viability, relative to undamaged cells, and increases the frequency of His+ recombinants containing translocations to 2.4 x 10(-4) (seven-fold), SCEs to 5.4 x 10(-4) (five-fold), and inversions to 1.8 x 10(-3) (six-fold). Compared to a haploids, DSB-stimulated frequencies in a/alpha haploids were three-fold higher for translocations, two-fold higher for SCEs, and ten-fold higher for inversions; however DSB-induced lethality was greater in a/alpha haploids. Compared to aa diploids, DSB-stimulated frequencies of translocations and viability after chromosome cleavage were greater in a/alpha diploids. We suggest that heterozygosity at MAT may elevate the frequency of DSB-initiated reciprocal exchange events in both haploid and diploid cells, but may only increase viability after chromosome cleavage in diploid cells.

A role for RAD51 in the generation of immunoglobulin gene diversity in rabbits.

Ig VDJ genes in rabbit somatically diversify by both hyperpointmutation and gene conversion. To elucidate the mechanism of gene conversion of IgH genes, we cloned a rabbit homologue of RAD51, a gene involved in gene conversion in Saccharomyces cerevisiae (yeast), and tested whether it could complement a yeast rad51 mutant deficient in recombination repair. We found that rabbit RAD51 partially complemented the defect in switching mating types by gene conversion as well as in DNA double-strand break repair after gamma-irradiation. Further, by Western blot analysis, we found that levels of Rad51 were higher in appendix-derived B lymphocytes of 6-wk-old rabbits, a time at which IgH genes diversify by somatic gene conversion. We suggest that Rad51 is involved in somatic gene conversion of rabbit Ig genes.

The Saccharomyces cerevisiae RAD9 checkpoint reduces the DNA damage-associated stimulation of directed translocations.

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-delta5' and trp1::his3-delta3'::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 G2-M checkpoint by demonstrating that if rad9 mutants were arrested in G2 before irradiation, the numbers both of UV- and gamma-ray-stimulated recombinants were reduced. The importance of G2 arrest in DNA damage-induced 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 G2-M checkpoint may reduce the frequency of DNA damage-induced translocations is by channeling the repair of double-strand breaks into SCE.

Genotoxicity of aflatoxin B1: evidence for a recombination-mediated mechanism in Saccharomyces cerevisiae.

The potent liver carcinogen aflatoxin B1 (AFB1) is metabolized by cytochrome P450 to the mutagenic epoxide. We have observed that activated AFB1 also strongly induced mitotic recombination in the yeast Saccharomyces cerevisiae. To compare the recombinogenicity of AFB1 to its mutagenicity, three metabolically competent S. cerevisiae strains have been constructed. The frequencies of induced recombinants resulting from gene conversion or chromosomal translocations were determined by different prototrophic selections using two strains, whereas the inducibility of forward mutations was determined by the frequency of drug resistance in the third strain. Human cytochrome P4501A1- (CYP1A) and NADPH-cytochrome P450-oxidoreductase cDNAs were expressed in the strains to ensure intracellular metabolism to the epoxide. Exposure of the strains to AFB1 resulted in a 139- and 24-fold increase in the translocation and gene conversion frequencies, respectively, whereas the mutation frequency was increased only 3-fold. In contrast, benzo[a]pyrene-7,8-dihydrodiol and ethyl methanesulfonate induced mutation and mitotic recombination to similar degrees. We conclude that AFB1 exerted a strong recombinogenic, but only a weak mutagenic, effect. The recombinogenicity of AFB1 in yeast may indicate a mechanism for the high proportion of loss of heterozygosity that has been detected in AFB1-related human liver cancers.

Mating type regulates the radiation-associated stimulation of reciprocal translocation events in Saccharomyces cerevisiae.

Both ultraviolet (UV) and ionizing radiation were observed to stimulate mitotic, ectopic recombination between his3 recombinational substrates, generating reciprocal translocations in Saccharomyces cerevisiae (yeast). The stimulation was greatest in diploid strains competent for sporulation and depends upon both the ploidy of the strain and heterozygosity at the MATlocus. The difference in levels of stimulation between MATa/MAT alpha diploid and MAT alpha haploid strains increases when cells are exposed to higher levels of UV radiation (sevenfold at 150 J/m2), whereas when cells are exposed to higher levels of ionizing radiation (23.4 krad), only a twofold difference is observed. When the MAT alpha gene was introduced by DNA transformation into a MATa/mat alpha::LEU2+ diploid, the levels of radiation-induced ectopic recombination approach those obtained in a strain that is heterozygous at MAT. Conversely, when the MATa gene was introduced by DNA transformation into a MAT alpha haploid, no enhanced stimulation of ectopic recombination was observed when cells were irradiated with ionizing radiation but a threefold enhancement was observed when cells were irradiated with UV. The increase in radiation-stimulated ectopic recombination resulting from heterozygosity at MAT correlated with greater spontaneous ectopic recombination and higher levels of viability after irradiation. We suggest that MAT functions that have been previously shown to control the level of mitotic, allelic recombination (homolog recombination) also control the level of mitotic, radiation-stimulated ectopic recombination between short dispersed repetitive sequences on non-homologous chromosomes.

DNA-damaging agents stimulate the formation of directed reciprocal translocations in Saccharomyces cerevisiae.

DNA-damaging agents can stimulate the formation of directed reciprocal translocations in strains of Saccharomyces cerevisiae containing his3 recombinational substrates to generate chromosomal rearrangements. Such agents were compared with those that can stimulate sister-chromatid recombination. We show that chemicals and environmental agents that produce a variety of DNA lesions, including bulky adduct, thymidine dimers, interstrand cross-links, double-strand breaks alkylated bases, can stimulate recombination to yield reciprocal translocations. Of the agents tested, only the alkylating agents methyl methanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and a bifunctional agent that causes bulky DNA adducts, 4-nitroquinoline-N-oxide (4-NQO), significantly stimulate sister-chromatid recombination in our assay. Factors that contribute to the stimulation of interchromosomal recombination include strain genetic background and ploidy.

Direction of chromosome rearrangements in Saccharomyces cerevisiae by use of his3 recombinational substrates.

We used the his3 recombinational substrates (his3 fragments) to direct large interchromosomal (translocations) and intrachromosomal (deletions and tandem duplications) rearrangements in the yeast Saccharomyces cerevisiae. In strains completely deleted for the wild-type HIS3 gene, his3 fragments, one containing a deletion of 5' amino acid coding sequences and the other containing a deletion of 3' amino acid coding sequences, were first placed at preselected sites by homologous recombination. His+ revertants that arose via spontaneous mitotic recombination between the two his3 fragments were selected. This strategy was used to direct rearrangements in both RAD52+ and rad52 mutant strains. Translocations occurred in the RAD52+ genetic background and were characterized by orthogonal field alternating gel electrophoresis of yeast chromosomal DNA and by standard genetic techniques. An unexpected translocation was also identified in which HIS3 sequences were amplified. Two types of tandem duplications of the GAL(7, 10, 1) locus were also directed, and one type was not observed in rad52 mutants. Recombination mechanisms are discussed to account for these differences.

Recombinational substrates designed to study recombination between unique and repetitive sequences in vivo.

Three recombination events, reciprocal recombination, sister-chromatid recombination, and gene conversion, were studied using substrates designed in vitro. Each type of recombination event can be monitored at any chromosomal location. We have shown that sister-chromatid recombination is induced mitotically by DNA damaging agents, such as methyl methanesulfonate and gamma-rays, but is decreased mitotically in strains defective in rad52. Reciprocal recombination by which circular plasmids integrate into the genome is unaffected by rad52 defective alleles and occurs by a different recombination pathway. Mechanisms are suggested by which gene conversion between sister chromatids can generate chromosome rearrangements.