Specific Saccharomyces cerevisiae genes are expressed in response to DNA-damaging agents.

Ruby, Stephanie W.; Szostak, Jack W.

doi:10.1128/mcb.5.1.75

Cited by 155 publications

(67 citation statements)

References 53 publications

(40 reference statements)

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“…Because mRNA analysis for the RAD52 gene indicated that this gene is not induced in response to X-ray damage, we constructed a gene fusion with the 5' region of RAD52 to serve initially as a negative control for the induction of RAD54. It has already been observed that various DNAdamaging agents induce a specific set of mRNA transcripts (22) and cloned lacZ fusions (31). The fact that we see little or no induction of RAD52 constructs while we do see a clear induction of RAD54 confirms that DNA damage to cells induces a specific subset of genes and is not a general induction signal for all genes.…”

Section: Discussionsupporting

confidence: 76%

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

Cole¹,

Schild²,

Lovett³

et al. 1987

Mol. Cell. Biol.

133

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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...

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Section: Discussionsupporting

confidence: 76%

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

Cole¹,

Schild²,

Lovett³

et al. 1987

Mol. Cell. Biol.

133

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“…Yeast and bacteria were grown as described (6). 4NQO (Sigma) was prepared and stored as a stock solution at 0.5 mg/ml as described by Ruby and Szostak (17 (7,15,25). A second RAD2-lacZ fusion (pRF2002) utilized an EcoRV site in the RAD2 coding region and expressed more f3-galactosidase activity than did pRF2001.…”

Section: Methodsmentioning

confidence: 99%

“…A qualitative filter assay for 3-galactosidase (15) was used to screen for plasmids containing the E. coli lacZ gene in the correct transcriptional and translational alignments relative to the RAD genes. Quantitative assays for /3-galactosidase in liquid cultures of yeast were carried out with extracts prepared as described (17). Enzyme activity was measured by using O-nitrophenyl (3-Dgalactopyranoside or 4-methylumbelliferyl /3-D-galactoside as substrates.…”

Section: Methodsmentioning

confidence: 99%

“…Effect of 4NQO on Expression of (3-Galactosidase. To examine the effect of DNA damage on single-copy RADI, RAD2, and RAD3 integrated fusions, the UV mimetic agent 4NQO was used initially because it has been shown by others that several yeast DIN (damage inducible) genes of unknown function are efficiently induced by this chemical (17). Yeast cells transformed with the integrating plasmid pSR16 containing a DINI-lacZ fusion served as a positive control for induction and expressed 8-galactosidase at levels -50-fold higher than those observed in untreated cells (Table 1).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

A yeast excision-repair gene is inducible by DNA damaging agents.

Robinson¹,

Nicolet²,

Kalainov³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

108

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Plasmids containing various RAD-lacZ gene fusions were integrated into the chromosome of haploid yeast cells. These integrant strains were tested for expression of Escherichia coli P-galactosidase after treatment with agents that damage DNA or interfere with normal DNA replication. We did not observe induction of single-copy RADI-lacZ or RAD3-lacZ fusion genes under the experimental conditions used. However, exposure of cells containing an integrated RAD2-lacZ fusion gene to UV-radiation, y-radiation, 4-nitroquinoline 1-oxide, or nalidixic acid resulted in 4-to 6-fold enhanced expression of j3-galactosidase. Induction of the RAD2 gene after treatment of untransformed cells with 4-nitroquinoline 1-oxide was confirmed by direct examination of RAD2 mRNA. Lower levels of induction (=5O%) were observed after treatment of cells with other chemicals. Induction of the RAD2-lacZ fusion gene was also observed in cells transformed with single-copy and multicopy autonomously replicating plasmids.Mutations at a number of loci in the yeast Saccharomyces cerevisiae cause increased sensitivity to killing by UVradiation and reduced ability to excise pyrimidine dimers from DNA (see ref. 1). Defects in at least five of these genes (RADI, RAD2, RAD3, RAD4, and RADIO) result in the inability to carry out specific incision of DNA, an early event in nucleotide excision repair (2, 3). These results suggest that the products ofthese five RAD genes are required for incision and/or preincision events associated with excision repair of base damage from chromatin in yeast cells. A requirement for multiple proteins for the incision of damaged DNA also has been demonstrated in the prokaryote Escherichia coli (see ref. 4). There is good evidence that the genes encoding some of these proteins (uvrA, uvrB, and urvD) are part of the inducible SOS regulon that responds to DNA damage (see ref.4). The inducibility of the uvrC gene is controversial (5), and there is no evidence for enhanced expression of polA after exposure of cells to DNA damage.The RADI, RAD2, RAD3, and RADIO genes of S. cerevisiae have been isolated in this laboratory (6-9) and by others (10-14) by using molecular cloning techniques. In the present study we investigated the possible induction of some of these genes after DNA damage, using RAD-lacZ fusions that express E. coli P-galactosidase in yeast. After the integration of a RAD2-lacZ fusion gene into a Rad+ yeast strain, we observed a significant increase in the level of f-galactosidase in cells exposed to UV-radiation, t-radiation, 4-nitroquinoline 1-oxide (4NQO), or nalidixic acid. Using 4NQO treatment as an example, we show that this response is strictly dependent on active protein synthesis and that increased levels of RAD2 transcripts are observed after treatment of untransformed cells. Lower levels of induction were observed in cells treated with other agents that damage DNA or interfere with normal DNA replication. Induction of integrated single-copy RADI-lacZ or RAD3-lacZ genes was not observed under these exp...

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“…[41][42][43][44][45] This became clear in the late 1990s with the use of high-throughput microarray technologies. 46 Genome-wide expression analysis in yeast has revealed that in response to replication stress induced by methyl methane sulphonate (MMS), ionizing radiation (IR), hydroxyurea (HU) and UV radiation, hundreds of genes are either induced or repressed.…”

Section: Checkpoint-dependent Transcriptional Regulation In Yeastmentioning

confidence: 99%

The checkpoint transcriptional response: Make sure to turn it off once you are satisfied

Smolka¹,

Oliveira²,

Harris³

et al. 2012

Cell Cycle

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and r.debruin@ucl.uc.uk T he replication checkpoint signaling network monitors the presence of replication-induced lesions to DNA and coordinates an elaborate cellular response that includes ample transcriptional reprogramming. Recent work has established two major groups of replication stressinduced genes in Saccharomyces cerevisiae, the DNA damage response (DDR) genes and G 1 /S cell cycle (CC) genes. In both cases, transcriptional activation is mediated via checkpoint-dependent inhibition of a transcriptional repressor (Crt1 for DDR and Nrm1 for CC) that participates in negative feedback regulation. This repressor-mediated regulation enables transcription to be rapidly repressed once cells have dealt with the replication stress. The recent finding of a new class of CC genes, named "switch genes," further uncovers a mode of transcription regulation that prevents overexpression of replication stress induced genes during G 1 . Collectively, these findings highlight the need for mechanisms that tightly control replication stress-induced transcription, allowing rapid transcriptional activation during replication stress but also avoiding longterm hyperaccumulation of the induced protein product that may be detrimental to cell proliferation.

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Specific Saccharomyces cerevisiae genes are expressed in response to DNA-damaging agents.

Cited by 155 publications

References 53 publications

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

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

A yeast excision-repair gene is inducible by DNA damaging agents.

The checkpoint transcriptional response: Make sure to turn it off once you are satisfied

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