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

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 beta-galactosidase gene. Yeast transformants with either an integrated or an autonomously replicating plasmid containing these fusions expressed beta-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 agreement with previous mRNA analyses. When MATa cells were arrested in G1 with alpha-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 GAL1 promoter, we showed that double-strand breaks alone are sufficient in vivo for induction of RAD54.     

A common element involved in transcriptional regulation of two DNA alkylation repair genes (MAG and MGT1) of Saccharomyces cerevisiae.

The Saccharomyces cerevisiae MAG gene encodes a 3-methyladenine DNA glycosylase that protects cells from killing by alkylating agents. MAG mRNA levels are induced not only by alkylating agents but also by DNA-damaging agents that do not produce alkylated DNA. We constructed a MAG-lacZ gene fusion to help identify the cis-acting promoter elements involved in regulating MAG expression. Deletion analysis defined the presence of one upstream activating sequence and one upstream repressing sequence (URS) and suggested the presence of a second URS. One of the MAG URS elements matches a decamer consensus sequence present in the promoters of 11 other S. cerevisiae DNA repair and metabolism genes, including the MGT1 gene, which encodes an O6-methylguanine DNA repair methyltransferase. Two proteins of 26 and 39 kDa bind specifically to the MAG and MGT1 URS elements. We suggest that the URS-binding proteins may play an important role in the coordinate regulation of these S. cerevisiae DNA repair genes.     

Characterization of stress-responsive behavior in Pseudomonas aeruginosa PAO: isolation of Tn3-lacZYA fusions with novel damage-inducible (din) promoters.

Although the pervasive soil and water microorganism Pseudomonas aeruginosa demonstrates heightened sensitivity to UV radiation, this species possesses a recA gene that, based on structural and functional properties, could mediate a DNA damage-responsive regulon similar to the SOS regulon of Escherichia coli. To determine whether P. aeruginosa encodes such stress-inducible genes, the response of P. aeruginosa to DNA-damaging agents including far-UV radiation (UVC) and the quinolone antimicrobial agent norfloxacin was investigated by monitoring the expression of fusions linking P. aeruginosa promoters to a beta-galactosidase reporter gene. These fusions were obtained by Tn3-HoHoI insertional mutagenesis of a P. aeruginosa genomic library. Eight different damage-inducible (din) gene fusions were isolated which lack homology to the P. aeruginosa recA gene. Expression of the three gene fusions studied, dinA::lacZYA, dinB::lacZYA, and dinC::lacZYA, increased following UVC and quinolone exposure but not following heat shock. Similar to E. coli SOS genes, the din genes were induced to different extents and with dissimilar kinetics following UVC irradiation.     

Rapid and preferential activation of the c-jun gene during the mammalian UV response.

Exposure of mammalian cells to DNA-damaging agents leads to activation of a genetic response known as the UV response. Because several previously identified UV-inducible genes contain AP-1 binding sites within their promoters, we investigated the induction of AP-1 activity by DNA-damaging agents. We found that expression of both c-jun and c-fos, which encode proteins that participate in formation of the AP-1 complex, is rapidly induced by two different DNA-damaging agents: UV and H2O2. Interestingly, the c-jun gene is far more responsive to UV than any other immediate-early gene that was examined, including c-fos. Other jun and fos genes were only marginally affected by UV or H2O2. Furthermore, UV is a much more efficient inducer of c-jun than phorbol esters, the standard inducers of c-jun expression. This preferential response of the c-jun gene is mediated by its 5' control region and requires the TPA response element, suggesting that this element also serves as an early target for the signal transduction pathway elicited by DNA damage. Both UV and H2O2 lead to a long-lasting increase in AP-1 binding activity, suggesting that AP-1 may mediate the induction of other damage-inducible genes such as human collagenase.     

Characterization of a novel DNA damage-inducible gene of Saccharomyces cerevisiae, DIN7, which is a structural homolog of the RAD2 and RAD27 DNA repair genes

A number of DNA damage-inducible genes (DIN) have been identified in Saccharomyces cerevisiae. In the present study we describe isolation of a novel gene, Din7, the expression of which is induced by exposure of cells to UV light, MMS (methyl methanesulfonate) or HU (hydoxyurea). The DNA sequence of DIN7 was determined. By comparison of the predicted Din7 amino acid sequence with those in databases we found that it belongs to a family of proteins which includes S. cerevisiae Rad2 and its Schizosaccharomyces pombe and human homologs Rad13 and XPGC; S. cerevisiae Rad27 and its S. pombe homolog Rad2, and S. pombe Exo I. All these proteins are endowed with DNA nuclease activity and are known to play an important function in DNA repair. The strongest homology to Din7 was found with the Dhs1 protein of S.?cerevisiae, the function of which is essentially unknown. The expression of the DIN7 gene was studied in detail using a DIN7-lacZ fusion integrated into a chromosome. We show that the expression level of DIN7 rises during meiosis at a time nearly coincident with commitment to recombination. No inducibility of DIN7 was found after treatment with DNA-damaging agents of cells bearing the rad53-21 mutation. Surprisingly, a high basal level of DIN7 expression was found in strains in which the DUN1 gene was inactivated by transposon insertion. We suggest that a form of Dun1 may be a negative regulator of the DIN7 gene expression.     

Characterization of a novel DNA damage-inducible gene of Saccharomyces cerevisiae , DIN7 , which is a structural homolog of the RAD2 and RAD27 DNA repair genes

A number of DNA damage-inducible genes (DIN) have been identified in Saccharomyces cerevisiae. In the present study we describe isolation of a novel gene, Din7, the expression of which is induced by exposure of cells to UV light, MMS (methyl methanesulfonate) or HU (hydoxyurea). The DNA sequence of DIN7 was determined. By comparison of the predicted Din7 amino acid sequence with those in databases we found that it belongs to a family of proteins which includes S. cerevisiae Rad2 and its Schizosaccharomyces pombe and human homologs Rad13 and XPGC; S. cerevisiae Rad27 and its S. pombe homolog Rad2, and S. pombe Exo I. All these proteins are endowed with DNA nuclease activity and are known to play an important function in DNA repair. The strongest homology to Din7 was found with the Dhs1 protein of S.␣cerevisiae, the function of which is essentially unknown. The expression of the DIN7 gene was studied in detail using a DIN7-lacZ fusion integrated into a chromosome. We show that the expression level of DIN7 rises during meiosis at a time nearly coincident with commitment to recombination. No inducibility of DIN7 was found after treatment with DNA-damaging agents of cells bearing the rad53-21 mutation. Surprisingly, a high basal level of DIN7 expression was found in strains in which the DUN1 gene was inactivated by transposon insertion. We suggest that a form of Dun1 may be a negative regulator of the DIN7 gene expression. Received: 30 May 1996 / Accepted: 26 September 1996     

The muc genes of pKM101 are induced by DNA damage

A gene fusion was constructed in vitro that resulted in the synthesis of a hybrid protein consisting of the amino-terminal segment of the MucB protein of the mutagenesis-enhancing plasmid pKM101 joined to an enzymatically active carboxy-terminal segment of the beta-galactosidase protein. In strains bearing this fusion, beta-galactosidase activity was induced by UV radiation and other DNA-damaging agents. A genetic analysis of the regulation of expression of the phi (mucB'-lacZ') fusion was consistent with the LexA protein acting as the direct repressor of the mucB gene. Examination of the expression of the mucA and phi (mucB'-lacZ') gene products in maxicells in the presence and absence of a high-copy-number plasmid carrying the lexA+ gene demonstrated that lexA regulated both the mucA and mucB genes, thus supporting our conclusion that the two genes are organized in an operon with the mucA gene transcribed first. An analysis of the effects of the recA430(lexB30) mutation on muc expression led to the discovery of the differential ability of the recA430 gene product to induce expression of a dinB::Mu d1(Ap lac) fusion located on the chromosome and the same phi (dinB'-lacZ+) fusion cloned into plasmid pBR322. Models to account for the role of the recA430 allele on the expression of damage-inducible genes and on mutagenesis are discussed.     

Expression of DNA damage-inducible genes of Escherichia coli upon treatment with methylating, ethylating and propylating agents

Several alkylation-inducible genes have been identified by construction of Mu-d1 (Apr lac) fusions to genes whose expression is increased in response to alkylation treatment, but not UV treatment. We have examined the induction of 4 different alkylation-inducible genes by treatment with a variety of methylating and ethylating agents, and a propylating agent. We have compared the induction of the alkylation-inducible genes with the induction of the sulA gene, which is a component of the SOS response to DNA damage. We find that the Ada-regulated adaptive response genes (ada-alkB, alkA and aidB) are induced primarily in response to methylation treatment. The ada-independent aidC gene is induced upon treatment with agents that alkylate predominantly by SN1 nucleophilic attack. aidC induction occurs only when cells are not aerated during treatment. The SOS response, as indicated by sulA induction, is strongly induced by all types of alkylating agents used.     

Proteins induced by DNA-damaging agents in cultured Drosophila cells

In Drosophila cultured cells, the effects of several DNA-damaging agents on the expression of proteins were investigated. Poly(A+) RNA prepared from both untreated cells and cells treated with DNA-damaging agents was translated in vitro. The translation products were analyzed by two-dimensional electrophoresis. Methyl methanesulfonate, the most potent agent used, induced about 25 proteins, some new and some enhanced pre-existing proteins. Angelicin plus near UV irradiation, 4-nitroquinoline N-oxide and ethyl methanesulfonate were efficient inducers. Mitomycin C, UV irradiation and hydrogen peroxide were poor inducers, inducing only a few proteins at low levels. A tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, and a DNA gyrase inhibitor, nalidixic acid, also were used. In this system they were weak inducers of new proteins. Several of the new or enhanced proteins were common to several agents, but others were agent specific. The distribution of mutagen-induced proteins was compared with that of proteins induced in cells heated at 37 degrees C. Some of the proteins induced by DNA-damaging agents were found to overlap heat-shock proteins. These results suggest that there are sets of induced genes that are regulated differently.     

Measurement of in vivo expression of the recA gene of Escherichia coli by using lacZ gene fusions.

A recA-lacZ protein fusion was constructed in vivo by using bacteriophage Mu dII301(Ap lac). The fusion contained the promoter and first 47 codons of the recA mutant, as determined by DNA sequence analysis. The fusion was cloned and used to construct a recA-lacZ operon fusion at the same site within the recA gene. These fusions were introduced into the Escherichia coli chromosome at the lambda attachment site either as complete or cryptic lambda prophages. Synthesis of beta-galactosidase from these fusions was inducible by UV radiation. As the UV dose was increased, induction became slower and persisted for a longer period of time. At low doses of UV radiation, more beta-galactosidase was produced in a uvrA mutant than in a wild-type strain; however, at high doses, no induced synthesis of beta-galactosidase occurred in a uvrA mutant. recA+ strains carrying either the protein or operon fusion on a multicopy plasmid showed reduced survival after UV irradiation. This UV sensitivity was not exhibited by strains containing a single copy of either fusion, however; hence, the fusions provide a reliable measure of recA expression.     

A New Type of Fusion Analysis Applicable to Many Organisms: Protein Fusions to the URA3 Gene of Yeast

We have made constructs that join the promoter sequences and a portion of the coding region of the Saccharomyces cerevisiae HIS4 and GAL1 genes and the E. coli lacZ gene to the sixth codon of the S. cerevisiae URA3 gene (encodes orotidine-5'-phosphate (OMP) decarboxylase) to form three in frame protein fusions. In each case the fusion protein has OMP decarboxylase activity as assayed by complementation tests and this activity is properly regulated. A convenient cassette consisting of the URA3 segment plus some immediately proximal amino acids of HIS4C is available for making URA3 fusions to other proteins of interest. URA3 fusions offer several advantages over other systems for gene fusion analysis: the URA3 specified protein is small and cytosolic; genetic selections exist to identify mutants with either increased or decreased URA3 function in both yeast (S. cerevisiae and Schizosaccharomyces pombe) and bacteria (Escherichia coli and Salmonella typhimurium); and a sensitive OMP decarboxylase enzyme assay is available. Also, OMP decarboxylase activity is present in mammals, Drosophila and plants, so URA3 fusions may eventually be applicable in these other organisms as well.     

The mkaC virulence gene of the Salmonella serovar Typhimurium 96 kb plasmid encodes a transcriptional activator

The intracellular growth and virulence of Salmonella serovar Typhimurium for mice is dependent on a plasmid-borne gene cluster termed mka. We studied the regulatory interactions of the genes mkaA, mkaB, mkaC and mkaD using lacZ gene fusions. Complementation experiments with cloned DNA fragments encoding each of the four MKa proteins indicated that mkaC enhances the expression of beta-galactosidase from the mkaA-, mkaB- and mkaC-lacZ gene fusions in trans. An mkaD-lacZ fusion or mkaA-lacZ fusion that did not contain DNA proximal to mkaB was not inducible with MkaC, indicating that at least mkaB and mkaA are induced together as an operon. MkaC is thus the first virulence protein whose function has been resolved.     

Gene fusions with lacZ by duplication insertion in the radioresistant bacterium Deinococcus radiodurans.

Deinococcus radiodurans is the most-studied species of a eubacterial family characterized by extreme resistance to DNA damage. We have focused on developing molecular biological techniques to investigate the genetics of this organism. We report construction of lacZ gene fusions by a method involving both in vitro splicing and the natural transformation of D. radiodurans. Numerous fusion strains were identified by expression of beta-galactosidase. Among these fusion strains, several were inducible by exposure to the DNA-damaging agent mitomycin C, and four of the inducible fusion constructs were cloned in Escherichia coli. Hybridization studies indicate that one of the damage-inducible genes contains a sequence reiterated throughout the D. radiodurans chromosome. Survival measurements show that two of the fusion strains have increased sensitivity to mitomycin C, suggesting that the fusions within these strains inactivate repair functions.     

Induction of S.cerevisiae MAG 3-methyladenine DNA glycosylase transcript levels in response to DNA damage.

We previously showed that the expression of the Saccharomyces cerevisiae MAG 3-methyladenine (3MeA) DNA glycosylase gene, like that of the E. coli alkA 3MeA DNA glycosylase gene, is induced by alkylating agents. Here we show that the MAG induction mechanism differs from that of alkA, at least in part, because MAG mRNA levels are not only induced by alkylating agents but also by UV light and the UV-mimetic agent 4-nitroquinoline-1-oxide. Unlike some other yeast DNA-damage-inducible genes, MAG expression is not induced by heat shock. The S. cerevisiae MGT1 O6-methylguanine DNA methyltransferase is not involved in regulating MAG gene expression since MAG is efficiently induced in a methyltransferase deficient strain; similarly, MAG glycosylase deficient strains and four other methylmethane sulfonate sensitive strains were normal for alkylation-induced MAG gene expression. However, de novo protein synthesis is required to elevate MAG mRNA levels because MAG induction was abolished in the presence of cycloheximide. MAG mRNA levels were equally well induced in cycling and G1-arrested cells, suggesting that MAG induction is not simply due to a redistribution of cells into a part of the cell cycle which happens to express MAG at high levels, and that the inhibition of DNA synthesis does not act as the inducing signal.