Functional recA, lexA, umuD, umuC, polA, and polB genes are not required for the Escherichia coli UVM response

Abstract

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...

Requirement for Homologous Recombination Functions for Expression of the mutA Mistranslator tRNA-Induced Mutator Phenotype in Escherichia coli

“…In this assay, M13 ssDNA bearing a single sitespecific lesion (εC-ssDNA) is transfected into an appropriate strain, and the resulting progeny phage are analyzed for mutations at the εC site by a quantitative multiplex sequence analysis procedure summarized in Fig. 2C (16,17,19,21). The assay depends on limited elongation of a prelabeled primer to characteristic lengths, depending on the base replacing the lesion upon replication.…”

Role of mismatch repair in the Escherichia coli UVM response

“…1 also makes specific predictions regarding the survival of lesion-bearing M13 ssDNA. The mutagenic effects observed upon UVM induction are accompanied by a significant reactivation effect in which the survival of εC DNA in induced cells is enhanced an average of sixfold compared with that in uninduced cells (38,52). This enhanced survival is independent of known SOS mutagenesis genes and has been termed UVM reactivation to distinguish it from the previously recognized RecA-dependent effect known as Weigle reactivation.…”

“…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 several suspected carcinogens, such as vinyl chloride and ethyl carbamate (4,17), as well as by endogenous agents, such as lipid peroxidation end products (5,33).…”

“…εC, the model lesion used in our studies, is inflicted by metabolites of several suspected carcinogens, such as vinyl chloride and ethyl carbamate (4, 17), as well as by endogenous agents, such as lipid peroxidation end products (5, 33). Recent results from our laboratory have indicated that the UVM response modulates mutagenesis at 1,N 6 -ethenoadenine, another example of a nonpairing exocyclic DNA lesion, but not at O 6 -methylguanine, a mispairing lesion (43).To account for the UVM response, several model mechanisms based on either induction or transient depletion of a cellular activity in response to DNA damage have been proposed (34,38,51). An especially attractive hypothesis, the one tested here, is summarized in Fig.…”

“…To account for the UVM response, several model mechanisms based on either induction or transient depletion of a cellular activity in response to DNA damage have been proposed (34,38,51). An especially attractive hypothesis, the one tested here, is summarized in Fig.…”