Induction of transversion mutations in Escherichia coli by N-methyl-N''-nitro-N-nitrosoguanidine is SOS dependent.

Escherichia coli alkA mutants, which are deficient for an inducible DNA glycosylase, 3-methyladenine-DNA glycosylase II, are sensitive to mutagenesis by low doses of the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). As many as 90% of the alkA-dependent mutations induced by MNNG are also umuC+ dependent and thus are due to DNA lesions that are substrates for the mutagenic functions of the SOS response. A great number of these mutations are base substitutions at A . T sites, particularly A . T transversions. We discuss which DNA lesions may be responsible for these mutations. Our results show that the induction of 3-methyladenine-DNA glycosylase II, which occurs as part of the adaptive response to alkylating agents such as MNNG, significantly reduces the mutagenicity as well as the lethality of alkylation damage.     

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.     

Alkylating Agents Induce Uvm, a Reca-Independent Inducible Mutagenic Phenomenon in Escherichia Coli

Noninstructive DNA damage in Escherichia coli induces SOS functions hypothesized to be required for mutagenesis and translesion DNA synthesis at noncoding DNA lesions. We have recently demonstrated that in E. coli cells incapable of SOS induction, prior UV-irradiation nevertheless strongly enhances mutagenesis at a noninstructive lesion borne on M13 DNA. Here, we address the question whether this effect, named UVM for UV modulation of mutagenesis, can be induced by other DNA damaging agents. Exponentially growing δrecA cells were pretreated with alkylating agents before transfection with M13 single-stranded DNA bearing a site-specific ethenocytosine residue. Effect of cell pretreatment on survival of the transfected DNA was determined as transfection efficiency. Mutagenesis at the ethenocytosine site in pretreated or untreated cells was analyzed by multiplex DNA sequencing, a phenotype-independent technology. Our data show that 1-methyl-3-nitro-1-nitrosoguanidine, N-nitroso-N-methylurea and dimethylsulfate, but not methyl iodide, are potent inducers of UVM. Because alkylating agents induce the adaptive response to defend against DNA alkylation, we asked if the genes constituting the adaptive response are required for UVM. Our data show that MNNG induction of UVM is independent of ada, alkA and alkB genes and define UVM as an inducible mutagenic phenomenon distinct from the E. coli adaptive and SOS responses.     

Inhibition of induction of adaptive response by o-vanillin in Escherichia coli B

Mutations induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were strongly enhanced in the presence of o-vanillin in E. coli B. The enhancement was also observed in uvrA, umuC, recA, polA, or alkB mutants. This effect was lower in an alkA mutant, but was restored in an alkA umuC double mutant. By contrast, the enhancing effect was almost blocked in an ada and ada umuC double mutant. It was necessary to add simultaneously MNNG and o-vanillin to the growth medium. Further investigations were conducted on the induction of ada and umuC genes using ada'-lacZ' and umuC'-lacZ' plasmids. o-Vanillin suppressed the induction of the ada gene by MNNG treatment, but not that of the umuC gene. In fact expression of the umuC gene was induced by lower concentrations of MNNG in the presence of o-vanillin. The results suggest that o-vanillin inhibits induction of the adaptive response, and consequently, the MNNG-induced mutation frequency is increased due to unrepaired O6-methylguanine.     

Homologous recombination prevents methylation-induced toxicity in Escherichia coli

Methylating agents such as N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and methyl methane sulfonate (MMS) produce a wide variety of N- and O-methylated bases in DNA, some of which can block replication fork progression. Homologous recombination is a mechanism by which chromosome replication can proceed despite the presence of lesions. The two major recombination pathways, RecBCD and RecFOR, which repair double-strand breaks (DSBs) and single-strand gaps respectively, are needed to protect against toxicity with the RecBCD system being more important. We find that recombination-deficient cell lines, such as recBCD recF, and ruvC recG, are as sensitive to the cytotoxic effects of MMS and MNNG as the most base excision repair (BER)-deficient (alkA tag) isogenic mutant strain. Recombination and BER-deficient double mutants (alkA tag recBCD) were more sensitive to MNNG and MMS than the single mutants suggesting that homologous recombination and BER play essential independent roles. Cells deleted for the polA (DNA polymerase I) or priA (primosome) genes are as sensitive to MMS and MNNG as alkA tag bacteria. Our results suggest that the mechanism of cytotoxicity by alkylating agents includes the necessity for homologous recombination to repair DSBs and single-strand gaps produced by DNA replication at blocking lesions or single-strand nicks resulting from AP-endonuclease action.     

Inhibitory effect of cadmium and mercury ions on induction of the adaptive response in Escherichia coli

Cadmium and mercury ions inhibited the promotion of ada and alkA gene expression in the adaptive process induced by methylating agents such as N-methyl-N-nitrosourea (MNU), methyl methanesulfonate (MMS) and methyl iodide in Escherichia coli. In fact, the induction of O6-methylguanine-DNA methyl-transferase (MGTase) by MNU was suppressed in E. coli in the presence of these metal ions. These ions potentiated mutagenesis induced by methylating agents such as MNU and MMS, but not that induced by ethylating agents, UV irradiation, or N4-aminocytidine. These comutagenic effects were observed in wild-type and umuC36 strains of E. coli but not in the ada-5 strain, which is unable to induce the adaptive response. These results suggest that the comutagenic effects of Cd2+ and Hg2+ are due to inhibition of ada and alkA gene expression promoted by methylated MGTase.     

Induction of the alkA gene of Escherichia coli in gram-negative bacteria.

A broad-host-range plasmid containing a fusion of the alkA and lacZ genes of Escherichia coli was introduced into various aerobic and facultative gram-negative bacteria--33 species belonging to 19 genera--to study the induction of expression of the alkA gene by alkylating agents. The bacteria included species of the families Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Vibrionaceae, Neisseriaceae, Rhodospirillaceae, and Azotobacteraceae. Results obtained show that all bacteria tested, except Aeromonas hydrophila, Agrobacterium tumefaciens, Hafnia alvei, Rhizobium meliloti, Salmonella enteritidis, Xanthomonas campestris, and those of the genus Rhodobacter, are able to induce the alkA gene of E. coli in the presence of N-methyl-N'-nitro-N-nitrosoguanidine. All these data indicate that the adaptive response to alkylating agents is present in bacterial species of several families and that the Ada box sequence must be widely conserved.     

Cloning of Micrococcus luteus 3-methyladenine-DNA glycosylase genes in Escherichia coli

The 3-methyladenine-DNA glycosylase (m3ADG) excises 3-methyladenine (m3A) residues formed in DNA after treatment with alkylating agents. In Escherichia coli, the repair of this type of damage depends on the products of the genes tagA and/or alkA, which code for m3ADG I (20 kDa) and II (30 kDa), respectively. The tagA- and alkA--single mutants are sensitive to alkylating agents, the double mutant much more so. We have cloned two genes of Micrococcus luteus that can partly substitute the function of the E. coli tagA- and alkA- genes. An M. luteus genome bank was made by shotgun cloning of EcoRI + BamHI-digested DNA into pBR322. Two hybrid plasmids were identified that confer methylmethane sulfonate (MMS) resistance to the tagA- ada+ mutant and a capacity to reactivate MMS-treated bacteriophage lambda. Each hybrid plasmid directed the synthesis of 21-kDa m3ADG in E. coli tagA- ada-, which were not inhibited by 4 mM m3A. However, the restriction maps of the two cloned genes were different, and they showed no sequence homology as judged by the lack of cross hybridization.     

Mutagenesis and repair of DNA damage caused by nitrogen mustard, N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU), streptozotocin, and mitomycin C in E. coli

Cytotoxicity and mutagenesis by streptozotocin, BCNU, nitrogen mustard, and mitomycin C were evaluated in E. coli mutants deficient in SOS repair, SOS-mediated mutagenesis, the adaptive response, and mutants that engage in aberrant mismatch repair. The results demonstrate that premutagenic lesions are caused by nitrogen mustard, BCNU and streptozotocin that are not repaired by ada or recognized by umuDC. Further, recA mutants were hypomutable after exposure to nitrogen mustard, BCNU, and streptozotocin compared to wild type. With the exception of the monofunctional nitrosourea, streptozotocin, both recA and uvrA gene products contribute to the repair of DNA damage caused by the alkylating agents tested. In the case of streptozotocin, although recA mutants were more sensitive than wild type, uvrA mutants were not. Moreover, while ada and alkA E. coli mutants showed increased sensitivity to streptozotocin, they were not more sensitive to the other alkylating agents evaluated.     

Gene expression in E. coli after treatment with streptozotocin

Gene induction by the methylating agents streptozotocin (STZ), N-methyl-N-nitrosourea (MNU), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was evaluated in E. coli fusion mutants. These mutants have fusions of the lac operon to genes induced by treatment with sublethal levels of alkylating agents and were previously selected from random insertions of the Mu-dl (Apr lac) phage by screening for induction of beta-galactosidase activity in the presence of methyl methanesulfonate or MNNG. The results demonstrate that STZ differs from MNNG and MNU in failing to induce aidC expression. Further, expression of aidC after exposure to MNU and MNNG occurs only in nonaerated cultures; aeration blocks the induction. Induction of aidD, alkA, aidB, and sfiA expression occurs with all 3 agents although at markedly lower concentrations of MNNG and STZ compared to MNU. alkA and to a lesser extent aidD mutants of E. coli strains were more sensitive to these agents, while no differences were evident between wild-type and aidB or aidC fusion mutants.     

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.     

A straight correlation between mutagenic activity and beta-galactosidase activity induced by monofunctional alkylating agents.

Eight monofunctional alkylating agents were examined for their ability to induce mutation in Salmonella typhimurium. The assay was carried out in S. typhimurium TA100 with the preincubation method. The SN1-type agents were more mutagenic than the SN2-type ones; besides, methylating agents exerted more mutagenic activity than ethylating ones. Those responses in the reversion assay were quite similar to the results obtained previously with the beta-galactosidase assay in Escherichia coli CSH26/pMCP1000 (alkA'-lacZ') as to the induction of the adaptive response. A good correlation was found between mutagenic potency in the reverse mutation assay and inducing potency in the beta-galactosidase assay.     

Involvement of Escherichia coli DNA polymerase IV in tolerance of cytotoxic alkylating DNA lesions in vivo

Escherichia coli PolIV, a DNA polymerase capable of catalyzing synthesis past replication-blocking DNA lesions, belongs to the most ubiquitous branch of Y-family DNA polymerases. The goal of this study is to identify spontaneous DNA damage that is bypassed specifically and accurately by PolIV in vivo. We increased the amount of spontaneous DNA lesions using mutants deficient for different DNA repair pathways and measured mutation frequency in PolIV-proficient and -deficient backgrounds. We found that PolIV performs an error-free bypass of DNA damage that accumulates in the alkA tag genetic background. This result indicates that PolIV is involved in the error-free bypass of cytotoxic alkylating DNA lesions. When the amount of cytotoxic alkylating DNA lesions is increased by the treatment with chemical alkylating agents, PolIV is required for survival in an alkA tag-proficient genetic background as well. Our study, together with the reported involvement of the mammalian PolIV homolog, Polkappa, in similar activity, indicates that Y-family DNA polymerases from the DinB branch can be added to the list of evolutionarily conserved molecular mechanisms that counteract cytotoxic effects of DNA alkylation. This activity is of major biological relevance because alkylating agents are continuously produced endogenously in all living cells and are also present in the environment.     

Effect of heat shock on expression of proteins not involved in the heat-shock regulon.

The effect of heat shock on the expression of some genes of Escherichia coli was tested. To avoid side effects, promoters of the genes were fused to lacZ and their expression measured by the level of beta-galactosidase. The results show that expression of umuC, recA and polB, after induction of the SOS response, was somewhat higher in the heat-shocked than in the non-shocked cells, whereas expression of ada, alkB and alkA genes, after induction of the adaptive response, was about the same. Unexpectedly, it was found that expression of lacZ from its own promoter was drastically lowered in the heat-shocked cells. This effect, however, seems not to be dependent on the induction of heat-shock proteins.     

Induction of SOS and adaptive responses by alkylating agents in Escherichia coli mutants deficient in 3-methyladenine-DNA glycosylase activities

The induction of SOS and adaptive responses by alkylating agents was studied in Escherichia coli mutants tagA and alkA deficient in 3-methyladenine-DNA glycosylase activities. The SOS response was measured using an sfiA::lacZ operon fusion. The sfiA operon, in the double mutant tagA alkA, is induced at 5-50-fold lower concentrations of all tested methylating and ethylating compounds, as compared to the wild-type strain. In all cases, the tagA mutation, which inactivates the constitutive and specific 3-alkyladenine-DNA glycosylase I (TagI), sensitizes the strain to the SOS response. The sensitization effect of alkA mutation, which inactivates the inducible 3-alkyladenine-DNA glycosylase II (TagII), is observed under conditions which allow the induction of the adaptive response. We conclude that the persistence of 3-methyladenine and 3-ethyladenine residues in DNA most likely leads to the induction of the SOS functions. In contrast, the adaptive response, evaluated by O6-methylguanine-DNA methyltransferase activity in cell extracts, was not affected by either tagA or alkA mutations. The results suggest that the SOS and adaptive responses use different alkylation products as an inducing "signal". However, adaptation protein TagII inhibits the induction of the SOS response to some extent, due to its action at the level of signal production. Finally, we provide conditions to improve short-term bacterial tests for the detection of genotoxic alkylating agents.     

Expression and functions of adaptive response genes in Escherichia coli treated with mono- and bifunctional alkylating agents. Interference with SOS response]

The expression of genes belonging to the Ada regulon of Escherichia coli under the action of mono- and bifunctional alkylating agents--high-efficiency antitumor HMM, ACNU, and BCNU preparations--was studied. The functional specificity of the alkA, alkB, and aidB1 genes concerning both the structure and volume of DNA alkylation and the specificity of cell preadaptation was revealed. Additional experimental evidence for the role of the aidB1 gene as a unique "hazard gene", a component of the E. coli ada operon, was obtained. A phenomenon of positive interference between alternative SOS and Ada responses was observed for the first time upon gene expression.     

Cloning and characterization of the alkA gene of Escherichia coli that encodes 3-methyladenine DNA glycosylase II

By in vitro recombination we have constructed hybrid plasmids which can suppress the increased methylmethane sulfonate sensitivity caused by the alkA1 mutation in Escherichia coli. Since the cloned DNA fragment was mapped at 44 to 45 min of the E. coli K12 genetic map, an area where the alkA gene is located, we conclude that the cloned DNA fragment contains the alkA gene itself but not other gene(s) that suppresses the alkA mutation. Specific labeling of plasmid-encoded proteins by the maxicell method revealed that the alkA codes for a polypeptide whose molecular weight is about 30,000. When cells harboring the alkA+ plasmids were grown in the presence of low doses of a simple alkylating agent (adapted condition), the activity of 3-methyladenine DNA glycosylase II was increased. The enzyme activity was copurified with the Mr 30,000 polypeptide. These results indicate that the alkA gene codes for 3-methyladenine DNA glycosylase II. Taking advantage of overproduction of the alkA protein in adapted cells that harbor multicopy plasmids carrying the alkA+ gene, 3-methyladenine DNA glycosylase II has been purified to apparent physical homogeneity.