Role of Mismatch Repair in the Escherichia coli UVM Response

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Role of Mismatch Repair in the Escherichia coli UVM Response. HOLLY S. MURPHY, VASEEM A. PALEJWALA, M. SAYEEDUR RAHMAN, PAUL M. DUNMAN,.
JOURNAL OF BACTERIOLOGY, Dec. 1996, p. 6651–6657 0021-9193/96/$04.0010 Copyright q 1996, American Society for Microbiology

Vol. 178, No. 23

Role of Mismatch Repair in the Escherichia coli UVM Response HOLLY S. MURPHY, VASEEM A. PALEJWALA, M. SAYEEDUR RAHMAN, PAUL M. DUNMAN, GE WANG, AND M. ZAFRI HUMAYUN* Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, Newark, New Jersey 07103-2714 Received 17 July 1996/Accepted 25 September 1996

Mutagenesis at 3,N4-ethenocytosine («C), a nonpairing mutagenic lesion, is significantly enhanced in Escherichia coli cells pretreated with UV, alkylating agents, or H2O2. 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. nonpairing exocyclic DNA lesion, but not at O6-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. 1. It is assumed that highly mutagenic translesion DNA synthesis across the εC residue on M13 ssDNA occurs in both uninduced cells and UVM-induced cells. However, the products of translesion synthesis are subject to postreplicative mismatch repair that acts unequally on the three major products of translesion synthesis containing εC:T, εC:A, or εC:G “base pairs.” This repair activity (perhaps an N-glycosylase) favors εC:A and εC:T base pairs, not εC:G pairs, as substrates and ultimately creates a gap across the lesion, thus recycling the damaged DNA template. This recycling process is expected to enrich for εC:G products, thus effectively lowering the observed mutation frequency in uninduced cells. In UVM-induced cells, depletion of the mismatch repair activity attributable to chromosomal DNA damage results in the survival of all products of the initial round of translesion synthesis, thereby increasing the apparent mutation frequency. Another attractive feature of this hypothesis is that it can account for the enhanced survival of εC DNA in UVMinduced cells (UVM reactivation). Template recycling in uninduced cells sequesters a large fraction of εC DNA from replicating, whereas in induced cells there is no template recycling. Replication of all translesion synthesis products in UVM-induced cells accounts for enhanced survival as well as mutagenesis. Several postreplicative mismatch repair pathways in E. coli have previously been described. The methyl-directed mismatch repair system is an example of a long-patch repair pathway that generates repair tracts of up to 103 bp (50). This pathway is known to recognize all single-base mismatches, albeit with different efficiencies. The methylation state of the DNA at

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,N4-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 H2O2. 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). Recent results from our laboratory have indicated that the UVM response modulates mutagenesis at 1,N6-ethenoadenine, another example of a

* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, 185 South Orange Ave. MSB F-607, Newark, NJ 07103-2714. Phone: (201) 982-5217. Fax: (201) 982-3644. Electronic mail address: humayun @umdnj.edu. 6651

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FIG. 1. A model mechanism for the UVM response through the suppression of postreplicative mismatch repair of the products of translesion DNA synthesis. An unidentified mismatch repair enzyme that recognizes εC:A and εC:T base pairs (but not εC:G base pairs) acts to remove the normal base (A or T) inserted opposite the lesion. The refilling of the repair gap requires a new round of translesion synthesis. Such template recycling ultimately results in an enrichment (selection) for the εC:G pairs that are resistant to repair, as shown at bottom left. Template recycling also acts as an impediment to viral replication, resulting in lower levels of survival and lower levels of mutagenesis. In UVM-induced cells (bottom right), this enzyme activity is depleted by damage to the chromosomal DNA. Therefore, there is less template recycling, simultaneously accounting for increased levels of mutagenesis as well as increased levels of viral survival.

specific sites (59GATC) provides the signal for strand-specific repair (16, 22, 40). MutH, MutL, and MutS proteins, along with a helicase (UvrD), exonucleases VII and I, and DNA polymerase III holoenzyme, are required for this pathway (31). Short-patch repair characteristically produces repair tracts of up to 20 nucleotides (31). An example of short-patch repair in E. coli is the very-short-patch (VSP) pathway that acts on T:G mismatches within the sequence 59CCWGG (10, 31, 42). The enzyme Dcm methylates the internal cytosine residue in the 59CCWGG sequence (25). T:G mismatches resulting from the spontaneous deamination of 5-methylcytosine are repaired (20) by a process that requires the Vsr endonuclease and DNA polymerase I, with MutS and MutL proteins acting as inessential accessory factors (6, 13, 14, 19, 47). The MutY glycosylase is responsible for a second shortpatch pathway in which adenine is removed from A:G, A:C, and A:8-oxoguanine (8-oxoG) mismatches (1, 2, 26, 27, 29, 41, 49). The MutM glycosylase initiates the third short-patch pathway that acts on a variety of DNA lesions, including the ringopen form of guanine termed formamidopyrimidine (28, 48) and the oxidized base 8-oxoG (26, 27, 29). MutY, MutM, and other enzymes are members of a network of mechanisms that reduce mutagenesis from oxidatively damaged DNA (11). In this communication, we have addressed the question of whether any of the known mismatch repair activities in E. coli are required for the UVM response. MATERIALS AND METHODS Bacterial strains and plasmids. Table 1 lists the E. coli strains and plasmids used in this study. The repair-deficient strains used here were tested for the expected mutator phenotype by measuring the levels of spontaneously arising rifampin-resistant mutants (30). Similarly, plasmids bearing the indicated genes

were confirmed to suppress the mutator phenotypes of the corresponding repairdeficient strains (data not shown). Construction of M13 ssDNA bearing a site-specific «C lesion. Construction of the lesion-bearing vector has been described in detail elsewhere (34) and is modeled after the technology developed by Banerjee and coworkers (3). Cell pretreatment, transfection, and preparation of pooled progeny phage.

TABLE 1. E. coli strains and plasmids used in this study Strain or plasmid

Strains KH2 KH2R

Relevant genotype or description

D(lac-pro) trpE4777 D(srlR-recA)306::Tn10 (Tetr) in KH2 CJ251 F9 (pOX38::Cmr) GM4271 F9 mutS458::mini-Tn10 (Kanr) GW3773 mutH471::Tn5 (Kanr) GW3773F GW3773 with F9 from CJ251 D6432 mutL::Tn10 (Tetr) D6432F F9 from CJ251 in D6432 HSM100 pACYC184 in KH2R HSM200 pMQ339 in KH2R HSM300 pMQ348 in KH2R HSM400 pMQ341 in KH2R GM31 dcm-6 GM380 dcm-6 F9 CC104 (mutY) mutY::mini-Tn10 (Tetr) CC104 (mutM) mutM::mini-Tn10 (Tetr) Plasmids pACYC184 Tetr Cmr (vector) pMQ339 pACYC184 with mutL gene (Cmr) pMQ341 pACYC184 with mutS gene (Cmr) pMQ348 pACYC184 with mutH gene (Cmr)

Source (reference)

This laboratory (44) This laboratory (37) C. Joyce (15) M. Marinus (24a) P. Modrich (8) This laboratory P. Modrich (8) This laboratory This laboratory This laboratory This laboratory This laboratory M. Marinus (39) M. Marinus (39) J. Miller (28) J. Miller (28) M. M. M. M.

Marinus Marinus Marinus Marinus

(53) (53) (53) (53)

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VOL. 178, 1996 E. coli cells were grown to exponential phase (108 cells per ml) in Luria-Bertani medium at 378C with vigorous aeration. Cells were treated with N-methyl-N9nitro-N-nitrosoguanidine (MNNG) (0, 1, 5, or 10 mg/ml) for 10 min at 378C as previously described by Wang et al. (52). Treated cells were pelleted, washed with an equal volume of Luria-Bertani medium, and processed for transfection as previously described (34). KH2R cells were used as the indicator strain (52) for measuring infectious centers, as well as for progeny phage amplification for experiments with the D6432F (mutL) strain. Preparation of pooled progeny phage DNA was carried out as previously described (36). Quantitative multiplex DNA sequence analysis. The mutation frequency and specificity of εC were determined by multiplex sequence analysis, as described previously (34, 36) and summarized in Fig. 2.

RESULTS Experimental rationale. To test the hypothesis that the UVM response is mediated by depletion of a postreplicative mismatch repair enzyme (Fig. 1), we pretreated E. coli strains deficient in known mismatch repair pathways with MNNG, an agent known to effectively induce the UVM response. The pathways included in this study were methyl-directed mismatch repair, VSP repair, and the MutY- and MutM-mediated repair pathways. If depletion of one of these repair pathways is the mechanism for UVM, a mutant in the repair pathway should display UVM constitutively, i.e., there should be high levels of mutagenesis at εC in uninduced cells and little change in pretreated cells. Conversely, if the repair mechanism is not involved in UVM, normal induction should occur with low-level mutagenesis in untreated cells and high-level mutagenesis in treated cells. We also tested whether UVM is suppressed in E. coli strains that overproduce the methyl-directed repair proteins MutH, MutL, and MutS. If UVM is mediated by a depletion of one of these factors, then overexpression should suppress the UVM response by compensating for the depletion in induced cells. If the repair factors are not involved, normal UVM induction is expected in overexpressing cells. In addition to predictions concerning mutagenesis, the hypothesis depicted in Fig. 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. As noted above, a mismatch repair-depletion model can account for UVM reactivation if it is assumed that the availability of the repair pathway sequesters the DNA and thereby reduces the efficiency of replication. Depletion of this repair activity in induced cells removes this block to replication and accounts for the reactivation effect accompanying high-level mutagenesis. Therefore, the model predicts that in the absence of repair, constitutive UVM mutagenesis is accompanied by high-level viral survival. Conversely, in strains overexpressing repair proteins, suppression of UVM mutagenesis coincides with a loss of UVM reactivation. The predictions mentioned above were tested by transfecting M13 ssDNA constructs bearing a site-specific εC residue into untreated and MNNG-pretreated cells either proficient or deficient for the repair pathways mentioned above (Fig. 2). Cells were grown to exponential phase and treated with MNNG for 10 min at 378C, with subsequent removal of the inducing agent and processing to prepare transfection-competent cells (37, 52). Survival effects were determined by plating aliquots of transfected cells for infectious centers, and mutagenic effects were determined by a quantitative multiplex sequencing assay, as summarized in Fig. 2.

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UVM induction in E. coli strains defective for methyl-directed mismatch repair. E. coli GW3773F (mutH), D6432F (mutL), and GM4271F (mutS) were tested for UVM induction by carrying out two to four independent transfections, with progeny DNA from each transfection subjected to three sequencing assays. Figure 3 shows an example of the effects of MNNG pretreatment of an E. coli mutL strain deficient in methyl-directed mismatch repair, and Table 2 shows quantitative data for mutation frequency and specificity in uninduced and induced cells for three different mutants defective for this repair pathway. In uninduced mutL cells (Fig. 3A), the mutation frequency was about 12%, whereas MNNG pretreatment at 5 and 10 mg/ml resulted in increases in mutagenesis to 42 and 49%, respectively (Table 2). Similar experiments with mutH and mutS cells showed mutation frequencies under uninduced conditions of 8 and 15%, respectively (Table 2). However, pretreatment with MNNG caused significant increases in mutagenesis (35% for mutH and 73% for mutS). The predominant base pair substitution at εC in induced cells was a C3A transversion. These results suggest that the UVM response is inducible in cells defective for methyl-directed mismatch repair genes mutH, mutL, and mutS. UVM induction in E. coli strains that overproduce methyldirected mismatch repair proteins. The model predicts that overproduction of repair proteins should partially or fully suppress the UVM effect. An example of the antimutator effect of overproducing wild-type repair proteins is provided by the ability of multicopy plasmid clones to suppress the mutator phenotypes of mutS (53) and mutD5 (an allele of dnaQ), the gene encoding the proofreading epsilon subunit of DNA polymerase III (46). To test such a possibility, plasmids carrying the mutH, mutL, or mutS gene were introduced into KH2R (DrecA mutH1 mutL1 mutS1) cells and the resulting strains were tested for the UVM response. Figure 3B shows the gel pattern of a multiplex sequence assay of progeny phage from a mutS11 strain; Table 2 shows quantitative data for the same strain. The mutation frequency in uninduced cells was low but increased upon MNNG pretreatment. A similar, essentially normal UVM response was observed in cells overexpressing MutH or MutL (Table 2). Therefore, overexpression of mismatch repair protein MutH, MutL, or MutS does not appear to significantly suppress UVM, a result that is inconsistent with a repairdepletion model. UVM induction in a strain deficient in VSP repair. The VSP pathway is responsible for correcting T:G mismatches arising from spontaneous deamination at 5-methylcytosines within a specific sequence context. The required factors for this pathway include the Vsr endonuclease, MutH, MutL, and DNA polymerase I. Even though the sequence context of the εC lesion does not resemble the normal repair target, VSP repair is suspected to work with some efficiency at other sequences (12, 21). A possible role for VSP repair in UVM was tested by analyzing mutation fixation at εC in a strain deficient in VSP repair (39). This strain, GM380, has a polar mutation (dcm-6) that reduces the activities of both the Dcm methylase and the downstream gene (vsr) encoding the Vsr endonuclease. Figure 3C and Table 2 show that uninduced dcm-6 cells displayed low-level mutagenesis and that upon treatment with MNNG, mutagenesis increased to approximately 50%. These results are supported by the previous observation that MutH and MutL, proteins required for methyl-directed mismatch repair as well as for a facilitating role in VSP repair, appear not to be required for UVM. Therefore, depletion of VSP enzymes is unlikely to account for the UVM response. UVM effects in strains defective for the N-glycosylase activities MutY and MutM. MutY and MutM are involved in a

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FIG. 2. The experimental system. Cells in mid-log phase were exposed to MNNG for 10 min, with subsequent removal of MNNG by cell pelleting and washing steps. Cells were transfected with M13 ssDNA bearing an εC residue (structure in inset at top right). The construction of M13 ssDNA bearing a site-specific εC residue has been described in detail previously (34). Aliquots of transfected cells were plated for infectious centers (ic) to measure M13 viral DNA survival, and the remainder was used to obtain pooled progeny phage DNA, which was subjected to multiplex sequence analysis based on the strategy depicted on the right and described in detail elsewhere (34, 35). The DNA sequence of the immediate sequence context of the lesion within the construct is shown (X 5 εC). A 59 32P (p)-end-labeled 19-mer primer (0.1 pmol) was annealed to the pooled progeny phage DNA (0.2 pmol) and was elongated in the presence of dGTP (1 mM), dCTP (1 mM), and ddTTP (10 mM) by T7 DNA polymerase devoid of 39359 exonuclease activity (Sequenase 2.0; U.S. Biochemicals). The omission of dATP and the substitution of ddTTP for dTTP resulted in the termination of all DNA synthesis after the insertion of a few nucleotides, with the length of the termination product depending on the sequence of the template as shown. These elongation products were separated by high-resolution denaturing gel electrophoresis, and computing densitometry was used to determine the mutation frequency and specificity from the normalized signal in 21- (C3T), 22- (C3A), 23- (21 nucleotide [nt]), and 24-mer (wild-type [WT]) bands. All elongation assays included a set of standard DNA mixes to monitor the sequencing reaction. Data are averages of at least six individual elongation reactions with DNA obtained from at least two independent transfections.

complex pathway that repairs oxidatively damaged DNA. MutY recognizes A:G and A:8-oxoG mispairs and removes the normal base, adenine, while MutM removes the modified base (8-oxoG) from DNA. Figure 3D and Table 2 show that the level of mutagenesis in uninduced cells was low; MNNG pretreatment resulted in a dose-dependent increase in mutagenesis. Therefore, these glycosylase genes do not appear to have a significant effect on UVM induction. UVM reactivation. UVM reactivation describes the enhancement in the survival of εC-bearing ssDNA in MNNGpretreated cells compared with that in untreated cells. The survival data in Table 2 show that significant (two- to eightfold)

UVM reactivation was observed for all the strains tested. Therefore, neither the loss of the tested repair activities nor the overexpression of specific repair proteins appears to interfere with UVM reactivation, supporting the conclusion that known E. coli mismatch repair activities are not required for the UVM response. DISCUSSION The idea that a transient mutator effect can result from depletion of mismatch repair in E. coli is attractive for a number of reasons. Saturation of mismatch repair is thought to

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the strong mutator phenotype associated with retron DNA expression was attributed to depletion of mismatch repair because it was suppressed by plasmids (the same plasmids used in this study) carrying genes for MutH, MutL, and MutS proteins (23). The data here, however, show an apparently normal UVM response in the presence of plasmids overexpressing MutH, MutL, or MutS. This observation supports the idea that methyl-directed mismatch repair depletion by itself cannot be the mechanism underlying UVM. The VSP repair pathway is a highly specialized repair pathway that repairs T:G mismatches to reduce C3T transitions at a consensus sequence related to the Dcm recognition sequence (59CCWGG). It is not known whether this pathway can recognize mispairs containing DNA damage, but it is clear that the loss of this pathway does not significantly alter the UVM response. Similarly, the observation of essentially normal UVM responses in mutY and mutM mutants indicates that they are

TABLE 2. Effects of MNNG pretreatment of E. coli strains on mutagenesis at an εC lesion on transfected M13 ssDNA FIG. 3. Examples of multiplex analyses of mutation fixation at an εC residue borne on M13 ssDNA transfected into uninduced or MNNG-induced E. coli strains. (A) Cells deficient for mutL; (B) mutS-overexpressing cells (HSM400); (C) dcm-6 cells deficient for VSP repair; (D) mutM cells deficient for Fpg glycosylase. Exponentially growing (optical density at 600 nm 5 0.35) cells were treated with MNNG (0, 1, 5, or 10 mg/ml) for 10 min at 378C, with subsequent cell pelleting and washing steps. Cells were made competent and transfected with M13 ssDNA bearing a site-specific εC lesion. To measure survival effects, two 0.1-ml aliquots of the transfection mix were plated for infectious centers. The remainder of the transfection mix was used to prepare pooled progeny phage DNA as previously described (36) and was subjected to multiplex sequence analysis. The DNA elongation products were fractionated on 16% polyacrylamide–8 M urea gels before autoradiography. The lengths of the major elongation products (21-, 22-, and 24-mers) are indicated. In uninduced cells (0 MNNG), the level of mutagenesis at εC was low (barely visible mutant bands). The level of mutagenesis was clearly increased in MNNG-pretreated cells, as indicated by the dose-responsive increase in the mutant fraction (C3T [21-mer] and C3A [22-mer]) and the corresponding decrease in the wild-type fraction (24-mer).

occur in the mutator strain (mutD5) in which the polymerase III holoenzyme is defective for proofreading (46). Overreplication of ss retron DNA bearing mismatches in intrastrand secondary structures confers a strong mutator phenotype in E. coli, suggesting that the phenotype results from a depletion of mismatch repair (24). As noted earlier, a mismatch repairdepletion model can also provide a satisfactory explanation for the reactivation effect that accompanies the mutagenic effects. Nevertheless, the experiments described here demonstrate that all strains deficient for methyl-directed mismatch repair, VSP repair, and the glycosylase activities MutY and MutM display normal UVM induction. It is known that cells with mutated mutH, mutL, and mutS genes have high-level spontaneous background mutagenesis and display increases in base pair substitutions (18, 45). Mutation frequencies in uninduced KH2R (DrecA mutH1 mutL1 mutS1) cells are 3 to 7% (34, 38, 52), whereas they are 10 to 15% in mutL and mutS cells. It is possible that mismatch repair contributes to error avoidance in uninduced cells; however, the data here demonstrate that loss of the repair activity by itself cannot account for the UVM response, which remains significantly inducible in cells deficient for methyl-directed mismatch repair. Depletion of methyl-directed mismatch repair is believed to be responsible for the extreme mutator phenotype of mutD5 cells because the phenotype is significantly alleviated by the presence of plasmids expressing MutL or MutH (46). Similarly,

Strain (genotype)a

GW3773F (mutH)

D6432F (mutL)

GM4271 (mutS)

HSM100/pACYC184 HSM300 (mutH11) HSM200 (mutL11) HSM400 (mutS11)

GM380 (dcm-6)

CC104 (mutY)

CC104 (mutM)

MNNG preM13 treatment survival b (mg/ml) (ic/50 ng)c

0 5 10 0 5 10 0 5 10 0 5 10 0 5 10 0 5 10 0 5 10 0 5 10 0 5 10 0 5 10

70 568 350 335 870 413 285 733 905 388 2,405 1,790 325 1,053 1,508 443 1,070 1,258 445 2,335 1,568 118 563 610 718 2,520 3,348 218 375 350

Mean frequency (%) of mutation at εC 6 SDd Total

C3T

C3A

868 28 6 3 35 6 5 12 6 4 42 6 7 49 6 10 15 6 3 51 6 6 73 6 5 862 56 6 2 64 6 1 14 6 1 34 6 10 41 6 2 11 6 3 35 6 2 46 6 2 13 6 2 44 6 14 69 6 13 862 46 6 5 48 6 5 13 6 5 42 6 4 62 6 4 16 6 4 59 6 11 71 6 6

464 10 6 1 462 261 261 363 463 18 6 6 17 6 5 260 11 6 0 961 564 461 261 260 361 662 863 963 10 6 3 261 662 563 863 763 10 6 2 10 6 2 19 6 6 11 6 3

464 18 6 2 31 6 6 10 6 5 40 6 8 47 6 11 11 6 2 33 6 1 56 6 8 662 45 6 2 55 6 1 963 29 6 10 38 6 2 963 31 6 2 40 6 1 562 36 6 11 59 6 11 661 40 6 6 43 6 3 662 35 6 6 52 6 5 662 40 6 5 61 6 7

a mutL11, mutH11, and mutS11 strains have a normal chromosomal complement of all three (mutHSL) genes and carry a multicopy plasmid (pACYC184) bearing a functional copy of the indicated gene. b Cells grown to an A600 of 0.35 were pretreated with the indicated concentration of MNNG in Luria-Bertani medium at 378C for 10 min with vigorous aeration followed by cell pelleting and washing steps. Washed cells were then made competent for transfection. c Data are averages from two to four transfections, except the data for strain HSM100/pACYC184, which were obtained from a single transfection. ic, infectious centers. d Data were derived by analyzing progeny DNA pools obtained from two to four independent transfection experiments (one transfection for the control strain HSM100/pACYC184), with each pool subjected to three multiplex sequence assays; numbers are rounded to the nearest integer. The data in the total column are the sums of the frequencies of C3T and C3A substitutions, which together constitute the overwhelming majority of mutations induced at εC.

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not required for UVM. Finally, UVM reactivation was observed in all the strains tested, supporting the conclusion that depletion of known mismatch repair activities does not account for UVM. It is important to note, however, that these results do not rule out the possible involvement of an as-yet-unrecognized postreplicative mismatch repair pathway in E. coli. The data reported here support the notion that the UVM phenomenon may represent a previously unrecognized cellular response to DNA damage. Whether UVM is mediated by a novel mismatch repair pathway or by an altogether different mechanism, such as an alteration of replication fidelity in treated cells, is an interesting question that we hope to address in the future. ACKNOWLEDGMENTS We thank C. Joyce, J. H. Miller, M. Marinus, and P. Modrich for bacterial strains and plasmids and M. Marinus for helpful suggestions. This work was supported in part by grant CN-113 from the American Cancer Society. H.S.M. received partial salary support from a graduate student fellowship awarded by the N.J. Commission on Cancer Research. REFERENCES 1. Au, K. G., M. Cabrera, J. H. Miller, and P. Modrich. 1988. Escherichia coli mutY gene product is required for specific A-G to C-G mismatch correction. Proc. Natl. Acad. Sci. USA 85:9163–9166. 2. Au, K. G., S. Clark, J. H. Miller, and P. Modrich. 1989. Escherichia coli mutY gene encodes an adenine glycosylase active on G-A mispairs. Proc. Natl. Acad. Sci. USA 86:8877–8881. 3. Banerjee, S. K., R. B. Christensen, C. W. Lawrence, and J. E. LeClerc. 1988. Frequency and spectrum of mutations produced by a single cis-syn thyminethymine cyclobutane dimer in a single-stranded vector. Proc. Natl. Acad. Sci. USA 85:8141–8145. 4. Barbin, A., and H. Bartsch. 1986. Mutagenic and promutagenic properties of DNA adducts formed by vinyl chloride metabolites. Role Cyclic Nucleic Acid Adducts Carcinog. Mutagenesis 70:345–358. 5. Chen, H.-J. C., and F.-L. Chung. 1994. Formation of etheno adducts in reactions of enals via autooxidation. Chem. Res. Toxicol. 7:857–860. 6. Dzidic, S., and M. Radman. 1989. Genetic requirements for hyper-recombination by very short patch mismatch repair: involvement of Escherichia coli DNA polymerase I. Mol. Gen. Genet. 217:254–256. 7. Echols, H., and M. F. Goodman. 1991. Fidelity mechanisms in DNA replication. Annu. Rev. Biochem. 60:477–511. 8. Feng, W.-Y., E. Lee, and J. B. Hays. 1991. Recombinagenic processing of UV-light photoproducts in nonreplicating phage DNA by the Escherichia coli methyl-directed mismatch repair system. Genetics 129:1007–1020. 9. Friedberg, E. C., G. C. Walker, and W. Siede. 1995. DNA repair and mutagenesis. ASM Press, Washington, D.C. 10. Grilley, M., J. Holmes, B. Yashar, and P. Modrich. 1990. Mechanisms of DNA-mismatch correction. Mutat. Res. 236:253–267. 11. Grollman, A. P., and M. Moriya. 1993. Mutagenesis by 8-oxoguanine: an enemy within. Trends Genet. 9:246–249. 12. Hennecke, F., H. Kolmar, K. Brundl, and H.-J. Fritz. 1991. The vsr gene product of E. coli K-12 is a strand- and sequence-specific DNA mismatch endonuclease. Nature (London) 353:776–778. 13. Jones, M., R. Wagner, and M. Radman. 1987. Mismatch repair and recombination in E. coli. Cell 50:621–626. 14. Jones, M., R. Wagner, and M. Radman. 1987. Mismatch repair of deaminated 5-methylcytosine. J. Mol. Biol. 194:155–159. 15. Joyce, C. M., and N. D. Grindley. 1984. Method for determining whether a gene of Escherichia coli is essential: application to the polA gene. J. Bacteriol. 158:636–643. 16. Kramer, B., W. Kramer, and H.-J. Fritz. 1984. Different base/base mismatches are corrected with different efficiencies by the methyl-directed DNA mismatch-repair system of E. coli. Cell 38:879–887. 17. Leithauser, M. T., A. Liem, B. C. Stewart, E. C. Miller, and J. A. Miller. 1990. 1,N6-Ethenoadenosine formation, mutagenicity and murine tumor induction as indicators of the generation of an electrophilic epoxide metabolite of the closely related carcinogens ethyl carbamate (urethane) and vinyl carbamate. Carcinogenesis 11:463–473. 18. Leong, P.-M., H. C. Hsia, and J. H. Miller. 1986. Analysis of spontaneous base substitutions generated in mismatch-repair-deficient strains of Escherichia coli. J. Bacteriol. 168:412–416. 19. Lieb, M. 1987. Bacterial genes mutL, mutS, and dcm participate in repair of mismatches at 5-methylcytosine sites. J. Bacteriol. 169:5241–5246. 20. Lieb, M. 1991. Spontaneous mutation at a 5-methylcytosine hotspot is prevented by very short patch (VSP) mismatch repair. Genetics 128:23–27.

J. BACTERIOL. 21. Lieb, M., E. Allen, and D. Read. 1986. Very short patch mismatch repair in phage lambda: repair sites and length of repair tracts. Genetics 114:1041– 1060. 22. Lu, A.-L., S. Clark, and P. Modrich. 1983. Methyl-directed repair of DNA base-pair mismatches in vitro. Proc. Natl. Acad. Sci. USA 80:4639–4643. 23. Maas, W. K., C. Wang, T. Lima, A. Hach, and D. Lim. 1996. Multicopy single-stranded DNA of Escherichia coli enhances mutation and recombination frequencies by titrating MutS protein. Mol. Microbiol. 19:505–509. 24. Maas, W. K., C. Wang, T. Lima, G. Zubay, and D. Lim. 1994. Multicopy single-stranded DNAs with mismatched base pairs are mutagenic in Escherichia coli. Mol. Microbiol. 14:437–441. 24a.Marinus, M. Personal communication. 25. May, M. S., and S. Hattman. 1975. Deoxyribonucleic acid-cytosine methylation by host- and plasmid-controlled enzymes. J. Bacteriol. 122:129–138. 26. Michaels, M. L., C. Cruz, A. P. Grollman, and J. H. Miller. 1992. Evidence that MutY and MutM combine to prevent mutations by an oxidatively damaged form of guanine in DNA. Proc. Natl. Acad. Sci. USA 89:7022– 7025. 27. Michaels, M. L., and J. H. Miller. 1992. The GO system protects organisms from the mutagenic effect of the spontaneous lesion 8-hydroxyguanine (7,8dihydro-8-oxoguanine). J. Bacteriol. 174:6321–6325. 28. Michaels, M. L., L. Pham, C. Cruz, and J. H. Miller. 1991. MutM, a protein that prevents G:C3T:A transversions is formamidopyrimidine-DNA glycosylase. Nucleic Acids Res. 19:3629–3632. 29. Michaels, M. L., J. Tchou, A. P. Grollman, and J. H. Miller. 1992. A repair system for 8-oxo-7,8-dihydrodeoxyguanine. Biochemistry 31:10964–10968. 30. Miller, J. H. 1992. A short course in bacterial genetics: a laboratory manual and handbook for Escherichia coli and related bacteria. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 31. Modrich, P. 1991. Mechanisms and biological effects of mismatch repair. Annu. Rev. Genet. 25:229–253. 32. Murli, S., and G. C. Walker. 1993. SOS mutagenesis. Curr. Opin. Genet. Dev. 3:719–725. 33. Nair, J., A. Barbin, Y. Guichard, and H. Bartsch. 1995. 1,N6-Ethenodeoxyadenosine and 3,N4-ethenodeoxycytidine in liver DNA from humans and untreated rodents detected by immunoaffinity/32P-postlabelling. Carcinogenesis 16:613–617. 34. Palejwala, V. A., G. A. Pandya, O. S. Bhanot, J. J. Solomon, H. S. Murphy, P. M. Dunman, and M. Z. Humayun. 1994. UVM, an ultraviolet-inducible RecA-independent mutagenic phenomenon in Escherichia coli. J. Biol. Chem. 269:27433–27440. 35. Palejwala, V. A., R. W. Rzepka, and M. Z. Humayun. 1993. UV irradiation of Escherichia coli modulates mutagenesis at a site-specific ethenocytosine residue on M13 DNA. Evidence for an inducible recA-independent effect. Biochemistry 32:4112–4120. 36. Palejwala, V. A., R. W. Rzepka, D. Simha, and M. Z. Humayun. 1993. Quanititative multiplex sequence analysis of mutational hotpspots. Frequency and specificity of mutations induced by a site specific ethenocytosine in M13 viral DNA. Biochemistry 32:4105–4111. 37. Palejwala, V. A., D. Simha, and M. Z. Humayun. 1991. Mechanisms of mutagenesis by exocyclic DNA adducts. Transfection of M13 viral DNA bearing a site-specific adduct shows that ethenocytosine is a highly efficient RecAindependent mutagenic noninstructional lesion. Biochemistry 30:8736–8743. 38. Palejwala, V. A., G. Wang, H. S. Murphy, and M. Z. Humayun. 1995. Functional recA, lexA, umuD, umuC, polA, and polB genes are not required for the Escherichia coli UVM response. J. Bacteriol. 177:6041–6048. 39. Palmer, B. R., and M. G. Marinus. 1994. The dam and dcm strains of Escherichia coli—a review. Gene 143:1–12. 40. Pukkila, P. J., J. Peterson, G. Herman, P. Modrich, and M. Meselson. 1983. Effects of high levels of DNA adenine methylation on methyl-directed mismatch repair in Escherichia coli. Genetics 104:571–582. 41. Radicella, J. P., E. A. Clark, and M. S. Fox. 1988. Some mismatch repair activities in Escherichia coli. Proc. Natl. Acad. Sci. USA 85:9674–9678. 42. Radman, M., and R. Wagner. 1986. Mismatch repair in Escherichia coli. Annu. Rev. Genet. 20:523–538. 43. Rahman, M. S., P. M. Dunman, G. Wang, H. S. Murphy, and M. Z. Humayun. Effect of UVM induction on mutation fixation at nonpairing and mispairing DNA lesions. Mol. Microbiol., in press. 44. Sambamurti, K., J. Callahan, X. Luo, C. P. Perkins, J. S. Jacobsen, and M. Z. Humayun. 1988. Mechanisms of mutagenesis by a bulky DNA lesion at the guanine N7 position. Genetics 120:863–873. 45. Schaaper, R. M., and R. L. Dunn. 1987. Spectra of spontaneous mutations in Escherichia coli strains defective in mismatch correction: the nature of in vivo DNA replication errors. Proc. Natl. Acad. Sci. USA 84:6220–6224. 46. Schaaper, R. M., and M. Radman. 1989. The extreme mutator effect of Escherichia coli mutD5 results from saturation of mismatch repair by excessive DNA replication errors. EMBO J. 8:3511–3516. 47. Sohail, A., M. Lieb, M. Dar, and A. S. Bhagwat. 1990. A gene required for very short patch repair in Escherichia coli is adjacent to the DNA cytosine methylase gene. J. Bacteriol. 172:4214–4221. 48. Tchou, J., H. Kasai, S. Shibutani, M.-H. Chung, J. Laval, A. P. Grollman, and S. Nishimura. 1991. 8-Oxoguanine (8-hydroxyguanine) DNA glycosylase

VOL. 178, 1996 and its substrate specificity. Proc. Natl. Acad. Sci. USA 88:4690–4694. 49. Tsai-Wu, J.-J., J. P. Radicella, and A.-L. Lu. 1991. Nucleotide sequence of the Escherichia coli micA gene required for A/G-specific mismatch repair: identity of MicA and MutY. J. Bacteriol. 173:1902–1910. 50. Wagner, R. J., and M. Meselson. 1976. Repair tracts in mismatched DNA heteroduplexes. Proc. Natl. Acad. Sci. USA 73:4135–4139. 51. Wang, G., and M. Z. Humayun. 1996. Induction of the Escherichia coli UVM

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response by oxidative stress. Mol. Gen. Genet. 251:573–579. 52. Wang, G., V. A. Palejwala, P. M. Dunman, D. H. Aviv, H. S. Murphy, M. S. Rahman, and M. Z. Humayun. 1995. Alkylating agents induce UVM, a recA-independent inducible mutagenic phenomenon in Escherichia coli. Genetics 141:813–823. 53. Wu, T.-H., and M. G. Marinus. 1994. Dominant negative mutator mutations in the mutS gene of Escherichia coli. J. Bacteriol. 176:5393–5400.