Transfection of M13 single-stranded viral DNA bearing a 3,N 4-ethenocytosine lesion into Escherichia coli cells pretreated with UV results in a significant elevation of mutagenesis at the lesion site compared to that observed in untreated cells. This response, termed UVM, for UV modulation of mutagenesis, is induced by a variety of DNA-damaging agents and is distinct from known cellular responses to DNA damage, including the SOS response. This report describes our observation, as a part of our investigation of the UVM phenomenon, that E. coli cells bearing a mutA or mutC allele display a UVM-constitutive phenotype. These mutator alleles were recently mapped (M. M. Slupska, C. Baikalov, R. Lloyd, and J. H. Miller, Proc. Natl. Acad. Sci. USA 93:4380-4385, 1996) to the glyV (mutA) and glyW (mutC) tRNA genes. Each mutant allele was shown to arise by an identical mutation in the anticodon sequence such that the mutant tRNAs could, in principle, mistranslate aspartate codons in mRNA as glycine at a low level. Because a UVM-constitutive phenotype resulting from a mutation in a tRNA gene was unexpected, we undertook a series of experiments designed to test whether the phenotype was indeed mediated by the expression of mutant glycine tRNAs. We placed either a wild-type or a mutant glyV gene under the control of a heterologous inducible promoter on a plasmid vector. E. coli cells expressing the mutant glyV gene displayed all three of the following phenotypes: (i) missense suppression of a test allele, (ii) a mutator phenotype measured by mutation to rifampin resistance, and (iii) a UVM-constitutive phenotype. These phenotypes were not associated with cells expressing the wild-type glyV gene or with cells in which the mutant allele was present but was not transcriptionally induced. These observations provide strong support for the idea that expression of mutant tRNA can confer a mutator phenotype, including the UVMconstitutive phenotype observed in mutA and mutC cells. However, our data imply that low-level mistranslation of the epsilon subunit of polymerase III probably does not account for the observed UVM-constitutive phenotype. Our results also indicate that mutA ⌬recA double mutants display a normal UVM phenotype, suggesting that the mutA effect is recA dependent. The observations reported here raise a number of intriguing questions and raise the possibility that the UVM response is mediated through transient alteration of the replication environment.

The Escherichia coli UVM response is a recently described phenomenon in which pretreatment of cells with DNA-damaging agents such as UV or alkylating agents significantly enhances mutation fixation at a model mutagenic lesion (3,N 4 -ethenocytosine; C) borne on a transfected M13 single-stranded DNA genome. Since UVM is observed in ⌬recA cells in which SOS induction should not occur, UVM may represent a novel, SOS-independent, inducible response. Here, we have addressed two specific hypothetical mechanisms for UVM: (i) UVM results from a recA-independent pathway for the induction of SOS genes thought to play a role in induced mutagenesis, and (ii) UVM results from a polymerase switch in which M13 replication in treated cells is carried out by DNA polymerase I (or DNA polymerase II) instead of DNA polymerase III. To address these hypotheses, E. coli cells with known defects in recA, lexA, umuDC, polA, or polB were treated with UV or 1-methyl-3-nitro-1-nitrosoguanidine before transfection of M13 single-stranded DNA bearing a site-specific ethenocytosine lesion. Survival of the transfected DNA was measured as transfection efficiency, and mutagenesis at the C residue was analyzed by a quantitative multiplex DNA sequencing technology. Our results show that UVM is observable in ⌬recA cells, in lexA3 (noninducible SOS repressor) cells, in LexA-overproducing cells, and in ⌬umuDC cells. Furthermore, our data show that UVM induction occurs in the absence of detectable induction of dinD, an SOS gene. These results make it unlikely that UVM results from a recAindependent alternative induction pathway for SOS genes. Similarly, UVM is observed in polA (deficient in DNA polymerase I) and polB (deficient in DNA polymerase II) cells, suggesting that neither polymerase plays an indispensable role in UVM induction. Furthermore, our data show that the UVM response is accompanied by enhanced survival (UVM reactivation) of M13 DNA bearing C. The observation of UVM reactivation makes simple repair-suppression models for UVM less attractive and increases the plausibility of mechanisms operating at the level of base insertion. We hypothesize that noncoding lesions fall into two categories. The so-called SOS-dependent (class 1) lesions require SOS functions at the extension (bypass) step, whereas class 2 noncoding lesions do not. It is proposed that UVM, a previously unrecognized damage-inducible response, modulates base insertion at noncoding lesions.Efficient replication past certain DNA lesions depends on factors not required for the replication of normal template DNA. In Escherichia coli, the required additional factors are thought to be encoded by the member genes of the SOS regulon. According to the widely accepted SOS hypothesis, unrepaired DNA damage induces the SOS genes, ultimately resulting in enhanced levels of specific gene products that are proposed to transiently alter the replication machinery.The major features of the regulation of the SOS regulon are well described (20,45,50,65,66,69). Under normal conditions, transcript...

Mutagenesis at 3,N4 -ethenocytosine (C), a nonpairing mutagenic lesion, is significantly enhanced in Escherichia coli cells pretreated with UV, alkylating agents, or H 2 O 2 . This effect, termed UVM (for UV modulation of mutagenesis), is distinct from known DNA damage-inducible responses, such as the SOS response, the adaptive response to alkylating agents, or the oxyR-mediated response to oxidative agents. Here, we have addressed the hypothesis that UVM results from transient depletion of a mismatch repair activity that normally acts to reduce mutagenesis. To test whether the loss of mismatch repair activities results in the predicted constitutive UVM phenotype, E. coli cells defective for methyl-directed mismatch repair, for veryshort-patch repair, or for the N-glycosylase activities MutY and MutM were treated with the UVM-inducing agent 1-methyl-3-nitro-1-nitrosoguanidine, with subsequent transfection of M13 viral single-stranded DNA bearing a site-specific C lesion. Survival of the M13 DNA was measured as transfection efficiency, and mutation fixation at the lesion was characterized by multiplex sequencing technology. The results showed normal UVM induction patterns in all the repair-defective strains tested. In addition, normal UVM induction was observed in cells overexpressing MutH, MutL, or MutS. All strains displayed UVM reactivation, the term used to describe the increased survival of C-containing DNA in UVM-induced cells. Taken together, these results indicate that the UVM response is independent of known mismatch repair systems in E. coli and may thus represent a previously unrecognized misrepair or misreplication pathway.The term inducible mutagenesis has been used to describe the transient mutator phenotype observed in cells exposed to UV or other mutagens. In the well-studied Escherichia coli model, a set of inducible genes constituting the SOS response is presumed to mediate this phenotype. It is widely accepted that induced SOS proteins are required for facilitating translesion synthesis at blocks to replication created by DNA damage (7, 9, 32) and that this translesion synthesis is accomplished by lowering the fidelity of DNA replication, accounting for so-called SOS mutagenesis. However, the biochemical mechanism that facilitates the replication of damaged DNA remains undescribed.Our investigation of the mutagenic properties of the nonpairing lesion 3,N 4 -ethenocytosine (εC) has revealed the existence of a novel UV-inducible mutagenic response in E. coli, termed UVM (for UV modulation of mutagenesis) (34). The UVM effect is observed as a significant increase in mutagenesis at an εC residue borne on M13 viral single-stranded DNA (ssDNA) in E. coli cells pretreated with UV, alkylating agents, or H 2 O 2 . This response has been shown to be distinct from known DNA damage-inducible responses, such as the SOS response, the adaptive response to alkylating agents, or the oxyR-mediated response to oxidative agents (38,51,52). εC, the model lesion used in our studies, is inflicted by metabolites of se...

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SummaryThe Escherichia coli UVM (UV Modulation of mutagenesis) response is a DNA damage-inducible mutagenic pathway detected as significantly increased mutagenesis at 3,N 4 -ethenocytosine (1C) lesions borne on transfected single-stranded M13 vector DNA. All major classes of DNA-damaging agents can induce UVM, and the phenomenon is independent of previously characterized mutagenic responses in E. coli. To understand this phenomenon further, we set out to identify and characterize mutants in the UVM response. Screening a mutant bank of cells defective for 1-methyl-3-nitro-1-nitrosoguanidine-inducible genes revealed that defects in the recN gene cause a constitutive elevation of mutagenesis at 1C residues. In contrast to normal cells that show < 6% mutagenesis at 1C lesions, but < 60% upon UVM induction, recN-defective strains display approximately 50% mutagenesis at 1C lesion sites in untreated cells. However, the recN-mediated mutagenesis response was found to require the recA gene and the umuDC genes, and could be suppressed in the presence of a plasmid harbouring the SOS transcriptional repressor LexA. These results imply that recN cells are constitutively active for SOS mutagenesis functions. The observation that 1C mutagenesis is enhanced in recN cells confirms previous findings that mutagenesis at 1C can also be independently elevated by the SOS pathway.