RAD1, an excision repair gene of Saccharomyces cerevisiae, is also involved in recombination.

Abstract

The RADI gene of Saccharomyces cerevisiae is required for the incision step of excision repair of damaged DNA. In this paper, we report our observations on the effect of the RADI gene on genetic recombination.Mitotic intrachromosomal and interchromosomal recombination in RAD+, radi, rad52, and other rad mutant strains was examined. The radl deletion mutation and some radl point mutations reduced the frequency of intrachromosomal recombination of a his3 duplication, in which one his3 allele is deleted at the 3' end while the other his3 allele is deleted at the 5' end. Mutations in the other excision repair genes, RAD2, RAD3, and RAD4, did not lower recombination frequencies in the his3 duplication. As expected, recombination between the his3 deletion alleles in the duplication was reduced in the rad52 mutant. The frequency of HIS3+ recombinants fell synergistically in the radl rad52 double mutant, indicating that the RADI and RAD52 genes affect this recombination via different pathways. In contrast to the effect of mutations in the RAD52 gene, mutations in the RADI gene did not lower intrachromosomal and interchromosomal recombination between heteroalleles that carry point mutations rather than partial deletions; however, the radlA mutation did lower the frequency of integration of lnear plasmids and DNA fragments into homologous genomic sequences. We suggest that RADI plays a role in recombination after the formation of the recombinogenic substrate.In the yeast Saccharomyces cerevisiae, 10 genes, RAD1, RAD2, RAD3, RAD4, RAD7, RADIO, RAD14, RAD16, RAD23, and MMS19, are known to be involved in excision repair of DNA damaged by UV light and by other agents that deform the DNA helix. The radi, rad2, rad3, rad4, and radiO mutants are highly sensitive to UV light and to other DNA-damaging agents and are totally defective in the incision of damaged DNA (18,27,36). The mms19, rad7, radI4, radi6, and rad23 mutants are not as sensitive to DNAdamaging agents and differ in excision proficiency from each other (18,19). In their absolute requirement for incision, the RADI, RAD2, RAD3, RAD4, and RADIO genes of S. cerevisiae resemble the Escherichia coli uvrA, uvrB, and uvrC genes. The UvrABC enzyme complex of E. coli cuts the damaged strand at two sites, which are 7 to 8 nucleotides 5' and 3 to 4 nucleotides 3' from the damage (29, 37). The UvrD and Poll enzymes catalyze the release of the UvrABC enzyme complex from the damaged site (4, 10).In contrast to the E. coli uvrA, uvrB, and uvrC genes, which are required only for incision of damaged DNA, some of the yeast incision genes seem to be involved in other cellular processes as well. The RAD3 gene, in addition to its role in incision, is essential for cell viability; rad3 deletion mutants are inviable (8, 21), and various rad3 mutants that are defective only in the excision repair function or that are temperature sensitive for growth but proficient in DNA repair exist (22, 23; R. Schiestl and S. Prakash, unpublished observations). The RAD3 protein has been purified in our lab...

RAD10, an excision repair gene of Saccharomyces cerevisiae, is involved in the RAD1 pathway of mitotic recombination.

“…The effects of the RADIO gene on mitotic recombination are very similar to those of the RADI gene. Like the radlA mutation (34), the radiOA mutation lowered mitotic recombination synergistically in combination with the radS2A mutation; in contrast, recombination was reduced to the same level in the radiA radJOA double mutant as in the radiA and radJOA single mutants. These observations indicate that the RADI and RADIO genes function in the same pathway of mitotic recombination and that this pathway constitutes an alternate and competing pathway to the RADS2 recombination pathway.…”

“…The RAD52 gene, required for double-strand break repair and recombination (23), is also involved in this recombination. However, the RADI and RADS2 genes affect mitotic recombination via alternate pathways, since the formation of HIS3+ recombinants is decreased synergistically in the radlA rad52A double mutant (34). Based on the observation that the radiA mutation reduces the efficiency of integration of linear plasmids and DNA fragments into homologous genomic sequences, we have suggested that the action of RADI in recombination occurs after the formation of the recombinogenic substrate (34).…”

“…The cloned genes have also been used to construct genomic deletion mutations. Studies with these mutations have revealed that RAD3 is essential for cell viability (8,20) and that RADI functions in mitotic recombination (12,34). Thus, in contrast to Escherichia coli, in which the uvrA, uvrB, and uvrC genes are required only for the incision of UV-damaged DNA, some of the yeast excision repair genes affect other cellular processes.…”

“…Previously, we showed that the radlA mutation lowers the rate of mitotic intrachromosomal recombination of a his3 duplication in which one of the his3 alleles has a deletion at the 3' end and the other has a deletion at the 5' end (34). The RAD52 gene, required for double-strand break repair and recombination (23), is also involved in this recombination.…”

“…Plasmids YEplacl95, pDG95, and pSP56 have been described previously (34 reading frame (ORF) of these genes with the S. cerevisiae URA3 gene or another selectable gene by the one-step gene disruption method (32). The radlA and rad2A mutations were constructed as described previously (34).…”