Regulation of RAD54- and RAD52-lacZ gene fusions in Saccharomyces cerevisiae in response to DNA damage.

Abstract

The RAD52 and RAD54 genes in the yeast Saccharomyces cerevisiae are involved in both DNA repair and DNA recombination. RAD54 has recently been shown to be inducible by X-rays, while RAD52 is not. To further investigate the regulation of these genes, we constructed gene fusions using 5' regions upstream of the RAD52 and RAD54 genes and a 3'-terminal fragment of the Escherichia coli I-galactosidase gene. Yeast transformants with either an integrated or an autonomously replicating plasmid containing these fusions expressed ,I-galactosidase activity constitutively. In addition, the RAD54 gene fusion was inducible in both haploid and diploid cells in response to the DNA-damaging agents X-rays, UV light, and methyl methanesulfonate, but not in response to heat shock. The RAD52-lacZ gene fusion showed little or no induction in response to X-ray or UV radiation nor methyl methanesulfonate. Typical induction levels for RAD54 in cells exposed to such agents were from 3-to 12-fold, in good agreeement with previous mRNA analyses. When MATa cells were arrested in Gl with a-factor, RAD54 was still inducible after DNA damage, indicating that the observed induction is independent of the cell cycle. Using a yeast vector containing the EcoRI structural gene fused to the GAL) promoter, we showed that double-strand breaks alone are sufficient in vivo for induction of RAD54.The regulation of DNA repair and recombination in the procaryote Escherichia coli has been extensively studied. One result of these studies has been the elucidation of the inducible SOS repair pathway, a component of which, the recA gene produpt, appears to control a number of genes involved in both the repair of DNA lesions and general DNA recombination (for a review, see reference 38). The regulation of such activities in eucaryotic systems has not yet been thoroughly elucidated. In Saccharomyces cerevisiae, mutations in three separate epistasis groups have been demonstrated to cause sensitivity to DNA-damaging agents such as UV light, X-rays, and chemical mutagens (12,14). However, each of these groups has a different phenotypic response to different kinds of damage, leading to the hypothesis that each group is involved in a separate repair pathway (13,25). Some mutations in one of these epistasis groups, the RAD50-57 group of genes, have been shown to be not only sensitive to the effects of ionizing radiation, but also to block meiotic recombination and to reduce some types of mitotic recombination (16,27). For this reason, the repair mediated by the RAD50-57 pathway has been postulated to be recombinational repair. Mutations in RAD54 and RAD52, the genes whose regulation we describe here, have been shown to be blocked in double-strand break repair (6,20,28). Additionally, rad52 mutations block significant levels of recombination in meiosis and yield inviable spores (15, 16). Although rad54 mutations previously had been reported to have little if any effect on meiosis, deletion mutations of the RAD54 gene have recently been isolated and shown to decrease bot...

The Requirement for ATP Hydrolysis by Saccharomyces cerevisiae Rad51 Is Bypassed by Mating-Type Heterozygosity or RAD54 in High Copy

“…However, the expression of a RAD54-lacZ fusion is reported to be twofold higher in MATa/␣ diploids than MATa/a diploids (9). Therefore, we tested a high-copy-number plasmid expressing RAD54 for suppression of the ␥-ray sensitivity of the rad51-K191R strain.…”

Regulation of the Saccharomyces cerevisiae DNA repair gene RAD16

“…The graph represents the relative amount of RADJ6 mRNA in cells released from a factor immediately after UV irradiation (Li), in cells released from a factor 2 h after UV irradiation (0) or in cells maintained in the continuously presence of a factor (@). (5,19,20,23,25). We have shown that repair of the transcriptional silenced HMLa locus is absent in both radl6 and rad7 mutants (36).…”

“…In the yeast Saccharomyces cerevisiae >30 genes called RAD genes are known to be involved in DNA repair (1 1,12). Amongst the RAD genes the RAD2, RAD6, RAD7, RAD18, RAD23 and RAD54 genes have been reported to be inducible upon DNA damage and during meiosis (4,5,19,20,(23)(24)(25)(26). The RAD2, RAD7, RAD23 gene products are involved in nucleotide excision repair, RAD6 and RAD18 are required for induced mutagenesis and post-replication repair and RAD54 functions in the repair of double-strand breaks and recombination (12).…”

Functional Conservation of the Yeast and Arabidopsis RAD54-Like Genes

“…This defect prevents rad54D cells from growing on media containing MMS, a mutagen that damages DNA (Chlebowicz and Jachymczyk 1979). The rad54D mutants are also more sensitive to UV radiation than wild-type cells, because of the involvement of Rad54 in the repair of some UVdamaged DNA molecules (Cole et al 1987;KeszenmanPereyra 1990).…”