INTRODUCTION

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Autonomous organisms maintain a high level of replication fidelity through a variety of constitutive and inducible error avoidance mechanisms. However, in response to environmental and physiological stimuli, cells seem to have the ability to transiently reduce replication fidelity. The operation of these transient mutator pathways has broad implications for human health because of their potential impact on areas as diverse as cancer, aging, and emergence of resistant pathogenic organisms. The best-known example of a DNA damage-inducible mutagenic pathway is the Escherichia coli SOS response (for a review, see reference 4). The SOS response is mediated by the induced expression of some 20 member genes of a regulon that is normally transcriptionally repressed by the binding of the LexA protein to promoter sequences. DNA damage leads to replication arrest and generates a signal that activates the RecA protein (itself coded by an SOS gene) into the SOS-active RecA* (coprotease) form. The RecA* protein derepresses the SOS regulon by cleaving the LexA repressor protein and also assists in proteolytic activation of the UmuD protein to the SOS-active UmuD' form. It is believed that RecA, UmuD', and UmuC proteins alter the replication fidelity of the E. coli DNA polymerase III holoenzyme and accounts for the error-prone replication observed in cells subjected to DNA damage. Although the precise biochemical roles of the three SOS proteins have not been fully described, two recent reports indicate that the RecA and UmuD'C proteins may have biochemical functions as replication cofactors during translesion DNA synthesis (20, 27). Interestingly, overexpression of the SOS gene dinB was shown to confer a mutator phenotype in which mutagenesis was significantly elevated at apparently undamaged sites (8). Thus, dinB is implicated in at least some forms of untargeted mutagenesis accompanying SOS mutagenesis.

A second damage-inducible response, termed UVM (for UV modulation of mutagenesis), has been recently described. UVM is detected as increased mutation fixation at a site-specific 3,N⁴-ethenocytosine (C) lesion borne on transfected M13 viral single-stranded DNA (ssDNA) in cells pretreated with a variety of DNA-damaging agents (6, 14). This response is distinct from the SOS response in its genetic requirements because UVM does not require functional recA, umuD, and umuC genes and occurs in cells under conditions where the SOS functions are not induced (17). UVM appears to significantly affect mutagenesis at class 2 noninstructive lesions such as C and possibly 1,N⁶-ethenoadenine but not at mispairing lesions such as O⁶-methylguanine (19). The mechanisms underlying the UVM response are not known, but the preponderance of evidence points to the transient induction of an error-prone replication activity (12-14, 17).

In this study, we have compared the individual effects of the SOS and UVM pathways on mutation fixation at two noninstructive (C and abasic [AP] site) lesions so that we may begin an assessment of the impact of these pathways on induced mutagenesis. C is an exocyclic lesion induced by carcinogens such as vinyl chloride and ethyl carbamate but it is now recognized to be also induced by endogenous mutagens and may thus be part of the spontaneous DNA damage burden of the cell. Because the exocyclic amino nitrogen in C is linked to the ring nitrogen 3 by a two-carbon bridge, two of the three Watson-Crick pairing positions become unavailable. C is highly mutagenic and induces mostly base substitutions, with CA and CT mutations predominating (7). Although C has the in vitro template characteristics of a noninstructive DNA lesion (23), it is highly mutagenic in recA cells (16). In contrast, AP-site, the other model lesion included here, is a classic example of a recA-dependent (SOS-dependent) noninstructive lesion. A large number of spontaneous as well as damage-initiated mechanisms generate AP sites. M13 ssDNA bearing AP sites is very poorly replicated in host cells unless the host has been induced for SOS functions, indicating that these sites act as strong replication blocks (9). The mutational specificity of AP sites suggests an AP:N base insertion-bypass mechanism in which N = A > T/G >> C.

The data presented here indicate that both SOS and UVM pathways significantly, and independently, elevate mutagenesis at C. However, mutational specificity analyses show that the UVM effect predominates over the SOS effect when both pathways are induced. Mutagenesis at AP sites is strongly stimulated by the SOS pathway, with the UVM pathway causing a significant further enhanced mutagenesis for which SOS induction is a prerequisite.

	MATERIALS AND METHODS

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Strains. Bacterial and plasmid strains are listed in Table 1. Plasmid pSR1000 (Fig. 1; 6,383 bp), expressing umuD'C genes under the control of a single inducible lac-derived promoter, P_trc, was constructed by ligating three fragments: (i) a 4,443-bp EcoRI-NcoI fragment from the P_trc vector plasmid pSE380 (2), (ii) a 1,897-bp EcoRI-ClaI fragment from plasmid pEC42 (3) containing the entire umuC gene as well as a 3' part of the umuD gene, and (iii) a synthetic oligonucleotide duplex created by annealing a 45-mer (5'-CATGGGCTTTCCTTCACCGGCAGCAGATTACGTTGAACAGCGCAT) and a 43-mer (5'-CGATGCGCTGTTCAACGTAATCTGCTGCCGGTGAAGGAAAGCC). The synthetic duplex had ClaI- and NcoI-compatible ends and completed the 5' sequence of the umuD' gene that encodes the equivalent of the UmuD' protein. To place the recA gene downstream of umuD'C genes as a part of the same operon, plasmid pSR1000 was modified to remove the transcription termination sequence at the end of the umuC gene as follows. A 1,649-bp fragment that included the umuD'C region of pSR1000 plasmid was amplified by PCR by using the primers P1 (same sequence as the 45-mer shown above) and P2 (5'-GCGGGAGCGCTTTTcTCgaGCCGCTAT). The P2 primer included three mismatches (lowercase letters) to create a site for XhoI (underlined) 9 bp downstream of the umuC translational termination codon. The PCR product was cut with BglII and XhoI to obtain a 1,102-bp fragment (containing the 3' part of the umuC gene) that was ligated to a 4,695-bp BglII-XhoI fragment also from pSR1000 to generate plasmid pSR1007 (Fig. 1; 5,797 bp). The recA gene of E. coli KH₂ was amplified by PCR using a forward primer (5'-GCTTCAACAGAACtcgagGACTATCCGG) with five mismatches (lowercase letters) that inserted an XhoI site (underlined) 26 bp upstream of the Shine-Dalgarno sequence of the recA gene. The reverse primer (5'-CGACGGGATGTTGAactaGTCATGGCAT) with four mismatches (lowercase letters) was used to create an SpeI site (underlined) 71 bp downstream of the UAA stop codon of recA gene. The 1,207-bp PCR product was digested with XhoI and SpeI to obtain a 1,175-bp fragment containing a promoterless recA gene which was ligated with the 5,787-bp XhoI-SpeI fragment from plasmid pSR1007 to obtain pSR1718 (6,962 bp). Functional characterization of pSR1007 and pSR1718 is described elsewhere in the text.

View larger version (29K): [in this window] [in a new window]   FIG. 1.   Maps of plasmids used in this study, depicting major features and relevant restriction sites. For construction details, see Materials and Methods.

Construction of ssDNA bearing site-specific lesions. Briefly, phosphoramidite derivatives of C (14, 17) and AP (29) residues were used to introduce these lesions into 17-mer oligonucleotides by chemical synthesis. Appropriate procedures at the deprotection stage to conserve the chemical integrity of the C lesion were used as described previously (14, 17). The synthetic AP residues are known to be stable under conditions of oligonucleotide synthesis and were synthesized by Midland Certified Reagent Company (Midland, Tex.). All oligonucleotides were purified by a final step of electrophoresis on high-resolution (polyacrylamide-urea) gels and did not appear to have impurities at a detectable level. M13 ssDNA vectors bearing site-specific C (14, 17) or synthetic AP (29) lesions were prepared as previously described in detail and summarized in Fig. 2A. The constructed ssDNAs were denatured in the presence of an antiscaffold oligonucleotide immediately before transfection (17, 18, 29).

Transfection, survival effects, and mutational effects. Procedures for transfection of the DNA constructs, for measurement of survival effects, and for multiplex sequence analyses have been described previously (14, 15, 17) and are summarized in Fig. 2B and C and in the legend to Fig. 2.

View larger version (25K): [in this window] [in a new window]   FIG. 2.   Summary of procedures used for construction of M13 ssDNAs bearing lesions, for SOS or UVM induction, for transfection, and for multiplex sequence analyses. (A) Schematic representation of procedures for construction of M13 ssDNA molecules bearing site-specific lesions. Detailed methods have been described previously (17, 29). (B) Procedures used for SOS and/or UVM induction, transfection, and measurement of survival and mutagenic effects (for details of the methodology, see references 17 and 28). Briefly, cells grown in LB medium to mid-log phase (optical density at 600 nm of 0.35) in either the presence (for SOS induction) or absence of 1 mM IPTG were exposed to MNNG (for UVM induction; 10 µg/ml, final concentration) for 10 min at 37°C, followed by cell pelleting by centrifugation, washing by resuspension in LB medium, and then a second pelleting step. The washed cells were resuspended in 1/10 original volume of cold TSS solution (LB medium containing 10% [wt/vol] polyethylene glycol 3350, 5% [vol/vol] dimethyl sulfoxide, and 20 mM MgCl2) and were processed to render them transfection competent (16). ssDNA (about 50 ng) was transfected into 1 ml of competent cells, and two 0.1-ml aliquots were plated for infectious centers (ic) to determine effects of the lesion and the strain on the transfected DNA. The remainder of the cells were used to prepare pooled progeny phage ssDNA as described elsewhere (14). (C) Principles of multiplex sequence analysis as applied to analysis of mutagenesis at C and AP lesions. A prelabeled 19-mer primer is annealed to the pooled progeny phage ssDNA and allowed to elongate in the presence of dGTP, dCTP, and ddTTP. Depending on the base at position N, limit-elongation products of characteristic length are produced; these are fractionated on high-resolution gels and quantitated as described elsewhere (14, 15). In the case of C, CA transversions will yield a 22-mer and CT transitions will yield a 21-mer. C is also known to induce 1 nt deletions that will give rise to a 23-mer. Wild-type sequence will give rise to 24-mers (note that any CG transversions can also give rise to a 24-mer, but C does not induce CG mutations at appreciable levels [7, 16]). In the case of AP sites, APT, APA, 1 nt, and APG/C mutations will yield a 21-mer, a 22-mer, a 23-mer, and a 24-mer, respectively.

	RESULTS

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Experimental system. The experimental system used consists of transfecting M13 ssDNA bearing a site-specific C or AP site lesion into cells in which the SOS pathway, the UVM response, or both can be induced through appropriate genetic manipulation and pretreatment with a UVM-inducing agent. Survival effects of the lesion are determined as transfection efficiency, and mutation fixation at the lesion site is monitored by a multiplex sequence analysis technology. M13 ssDNA replication is known to proceed through two stages. In stage I, complementary (minus)-strand initiation occurs at a unique site (Ori) by the synthesis of a 20-nucleotide (nt)-long RNA primer by the host RNA polymerase. The primer is elongated around the genome by DNA polymerase III. Primer removal and gap filling are thought to be carried out by DNA polymerase I. Subsequent nick sealing by DNA ligase and negative supercoiling by DNA gyrase give rise to replicative form I (RF-I) DNA. Thus, mutation fixation is thought to occur during ssDNARF DNA (stage I) replication that depends exclusively on host replication proteins. In stage II, a rolling circle DNA replication mode that requires a phage-specified protein mediates both RFRF replication and RFssDNA replication.

Construction of E. coli strains in which expression of SOS mutagenesis proteins is uncoupled from DNA damage. While the SOS response is well characterized, and mutants defective for the SOS response are available, UVM-defective mutants are not yet available. Because DNA-damaging treatments can induce both pathways, we needed a means to allow for the expression of the SOS pathway without concomitant expression of the UVM response. To achieve this end, we opted for the strategy of placing expression of the SOS proteins under the control of a heterologous promoter rather than under the control of DNA damage-inducible SOS promoters. Plasmid pSR1718 (Fig. 1) was constructed by cloning the coding sequences for UmuD', UmuC, and RecA proteins under the control of a P_trc promoter in the vector pSE380 (2). A recA umuDC E. coli host cell bearing plasmid pSR1718 should express the three SOS proteins in response to the lac inducer isopropyl--D-thiogalactoside (IPTG) rather than to DNA damage.

recA umuDC

Effect of SOS and UVM pathways on survival of M13 ssDNA bearing site-specific C or AP lesions. To determine the individual effects of the SOS and UVM pathways on survival and mutagenesis at representative replication-blocking DNA lesions, we used the strategy summarized in Fig. 2A to construct M13 ssDNA genomes bearing single site-specific lesions. To induce the SOS pathway, cells were grown in the presence of IPTG; to induce the UVM pathway, cells were exposed for 10 min to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) at a concentration of 10 µg/ml. As summarized in Fig. 2B, induced cells were rendered transfection competent and were transfected with the appropriate ssDNA. Survival effects were measured as transfection efficiency (infectious centers [IC] per milliliter), and pooled progeny phage from each transfection were collected for analysis of mutation fixation. The data in Table 3 compare the effects of the induction of the SOS mutagenesis proteins (UmuD' and UmuC), the UVM pathway, or both on the survival of C-bearing ssDNA. In the absence of the induction of either pathway, survival is about 655 IC/ml. Survival increases approximately threefold by the induction of either the SOS mutagenesis pathway (umuD'C genes) or the UVM pathway. Interestingly, there is an additional twofold effect when both are induced, consistent with the simultaneous operation of two independent mechanisms. A similar pattern is observed in the recA umuDC strain expressing plasmid-borne umuD', umuC, and recA genes (Table 3). UVM or SOS induction elevates survival about twofold (Table 3; compare row 5 to rows 6 and 7), and induction of both elevates survival almost fourfold (compare rows 5 and 8). The UVM effect on survival does not require that the umuD, umuC, and recA genes be expressed at a basal level: it is observed in strain SR400, which is devoid of these genes (rows 9 and 10).

View this table: [in this window] [in a new window]   TABLE 3.   Effects of SOS and UVM pathways on mutagenesis at C

umuDC recA

Effect of SOS and UVM pathways on mutation fixation at C. The defining attribute of UVM is that its manifestation does not require functional recA, umuD, and umuC genes that are known to be required for SOS mutagenesis. To test whether the induction of the SOS pathway by itself can also affect mutation fixation at C, we carried out the experiments in which SOS mutagenesis gene expression was placed under heterologous promoter control. Figure 3A shows the basic elements of the UVM response in wild-type (KH2; lanes 1 and 2), recA (KH2R; lanes 3 and 4), and recA umuDC (SR400; lanes 5 and 6) strains. In uninduced cells, there is a low level of mutagenesis (little signal in 22-mer and 21-mer bands in comparison to the 24-mer in lanes 1, 3, and 5), whereas in UVM-induced cells there is a very high level of mutagenesis (lanes 2, 4, and 6).

View larger version (45K): [in this window] [in a new window]   FIG. 3.   Multiplex sequence analyses of mutagenesis at C (A and B) and AP (C) lesions under conditions where SOS, UVM, or both are induced under conditions summarized in the legend to Fig. 2. WT, wild type.

Effect of SOS and UVM pathways on base insertion opposite an AP site. The known genetic requirements for mutagenesis at AP sites indicate that SOS induction is necessary for mutation fixation opposite these classic noninstructive lesions. Because DNA damage can induce both SOS and UVM responses, it is interesting to assess the relative contributions of the SOS and UVM responses to mutation fixation at AP sites. Figure 3C, lane 1, shows that in the absence of UVM induction, a majority of mutational events in strain SR420 are 1 nt deletions (23-mer band), but base substitution events, such as APT (21 nt; product of an insertion of A opposite AP) and APC/G (AP:G and AP:C insertion events) are also visible. In quantitative terms (Table 4, row 1), deletions accounted for 74% of the bypass events, whereas APT (17%) and APC/G (8%) accounted for most of the remainder. Figure 3C (lane 2) and Table 4 (row 2) show that UVM induction causes a detectable redistribution of bypass events: deletions are apparently decreased to 51%, and there is an increase in APT mutations to 40% of the bypass events. Full induction of umuD' and umuC proteins (Fig. 3C, lane 3) dramatically increases base substitution mutations apparently at the expense of deletions: Table 4 (row 3) shows that APT events now account for 63%, with more modest increases in APC/G (12%) and APA (4%) events, but a drop in deletions to about 21%. UVM induction (Table 4, row 4) appears to accentuate this trend, with increases in APC/G mutations to 22% and APA mutations to 6% and a further decrease in deletions to 7%.

View larger version (37K): [in this window] [in a new window]   FIG. 4.   Number of insertion events at a site-specific AP site (A) or C (C) in M13 ssDNA transfected into E. coli SR440. The numbers of insertion events (from Tables 3 or 4) were normalized to ssDNA survival under each experimental condition by multiplying the fraction of represented by each type of insertion with the number of IC per milliliter (i.e., per transfection). For example, the AP:A fractions in strain SR440 under UVM SOS, UVM+ SOS, UVM SOS+, and UVM+ SOS+ conditions are, respectively, 0.496, 0.434, 0.766, and 0.726 (Table 4, fifth column, rows 5 to 8). Multiplying each fractional value with the corresponding survival value yields the following normalized numbers for AP:A insertion events: 258, 304, 1,259, and 2113. (B) Analysis of insertion events at AP sites similar to that in panel A except that data from SR400 (Table 4, rows 9 and 10) were used to calculate SOS UVM and SOS UVM+ conditions and data from SR440 were used for the UVM SOS+ and UVM+ SOS+ conditions. Thus, the AP:A fractions used for normalizing the number of events under UVM SOS, UVM+ SOS, UVM SOS+, and UVM+ SOS+ conditions were 0.024, 0.082, 0.766, and 0.726, respectively; the corresponding normalized numbers were 3, 8, 1,259, and 2,113. (D) Analysis of insertion events at C similar to that shown in panel C except that data from strain SR400 were used to represent the UVM SOS and UVM+ SOS conditions as described above.

	DISCUSSION

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In this report, we present the results of an analysis of the relative contributions of two genetically distinct DNA damage-inducible pathways to mutagenesis at two representative noninstructive DNA lesions. A prerequisite for this study has been the development of strains in which SOS, UVM, or both can be induced. Because of the lack of defined UVM-defective mutations, we chose the strategy of uncoupling SOS induction from DNA damage by expressing SOS mutagenesis genes under the control of a heterologous promoter. A similar strategy was previously used by Boudsocq et al., who placed the umuD' and umuC genes under the control of the pBAD promoter (1). Because of the subsequent report that expression of genes under pBAD control can be heterogeneous (22), we opted for the strategy of placing the SOS genes under the control of a lac-derived promoter. This strategy allows one to induce the SOS mutagenesis proteins by the simple means of adding IPTG to the growth medium. The data summarized in Table 2 confirm that SOS mutagenesis is IPTG inducible, as judged by the restoration of UV resistance and UV mutability to umuDC cells and to umuDC and recA cells by plasmids expressing the missing proteins under the control of the P_trc promoter. These data provide further confirmation to previous conclusions that expression of UmuD' and UmuC proteins is necessary and sufficient for UV mutagenesis, and they also confirm that constitutive levels of RecA protein are sufficient for SOS mutagenesis and that the third role of RecA in SOS mutagenesis can be satisfied with uninduced levels of normal (as opposed to RecA*) RecA protein (24, 26). In these strains, UVM is induced by a 10-min exposure of growing cells to MNNG at a final concentration of 10 µg/ml. This exposure condition is known to strongly induce the UVM but not the SOS response (17, 28). Exposure of IPTG-induced cells to MNNG should induce both SOS and UVM pathways.

The UVM response is defined by elevated mutagenesis at an C lesion (borne on a transfected M13 ssDNA genome) in cells subjected to DNA damage. This response has been shown to be independent of recA umuD and umuC genes and to be elicited by all major classes of DNA-damaging agents. The results presented in this report show that mutation fixation at C is not only elevated by the UVM response but also elevated independently by the SOS response. However, there is a difference in the specificity of mutations induced by the two pathways at C. The SOS pathway induces CT and CA mutations in about equal proportions, with perhaps a slight excess of the CT mutations. In contrast, UVM induction produces more CA mutations than CT mutations. When both pathways are induced, the total number of mutations is increased further, but a strong bias toward CA mutations that is a characteristic of UVM is maintained. Further analysis reveals that the apparent UVM dominance consists of both additive and competing effects (Fig. 4C and D): CA (C:T) mutations are elevated threefold by the combined actions of SOS and UVM pathways, whereas SOS-induced CT (C:A) mutations induced are reduced twofold by UVM.

A new insight to emerge from the AP site investigation is the possibility that the UVM response may contribute to mutagenesis at some SOS-dependent lesions. The data plotted in Fig. 4 show that in SOS-induced cells there is a consistent further increase in mutagenesis at AP sites upon UVM induction. The manifestation of this additive effect appears to require the full expression of SOS functions. Thus, our results show that the UVM response can affect mutagenesis at both class 2 (C) and at class 1 (AP site) lesions. Another interesting finding here is that cells appear to have a low but finite capacity for skip-bypass replication across AP sites such that targeted 1-bp deletions arise at a constant rate unaffected by the induction status of UVM or SOS responses. (A less likely possibility is that AP site oligonucleotide used in the construction of the ssDNA vector had a low-level contaminating sequence that was the equivalent of a 1 deletion). Increased base substitution mutagenesis at AP sites does not appear to occur at the expense of deletions in induced cells, as the data in Table 4 might suggest. Thus, most of the transfected AP-containing ssDNA apparently suffers replication arrest at the lesion site. A small but appreciable proportion of the arrested molecules are released into replication by a loop-out of the AP site so that the polymerase in effect skips across the lesion site, and this capability is constitutively expressed. The remainder of the arrested molecules simply perish in the absence of inducible factors.

It is interesting to compare our results with previous analyses of translesion DNA synthesis and mutagenesis at single AP sites borne on M13 ssDNA vectors (9, 10). Lawrence et al. (9) showed that very low levels of survival occurred in unirradiated cells, with survival increasing 10-fold in UV-irradiated SOS-proficient cells, results that are similar to ours (Table 4). Also, their observation that survival is not increased in SOS-defective cells even when subjected to DNA damage is consistent with the present work (Table 4, rows 9 and 10). However, by uncoupling SOS induction from DNA damage, we show here that DNA damage can increase translesion synthesis through an SOS-independent (UVM) pathway in SOS-induced cells (Table 4, rows 7 and 8). The mutation spectrum observed in two previous studies using a similar (but not identical) experimental system is also largely in agreement with the current data, in that predominantly A is inserted opposite AP sites, followed by other bases such as T and G (9, 10) in SOS-induced cells. However, our data show that a significant fraction of translesion synthesis in cells defective for SOS functions (SR400; Table 4, row 9) proceeds through a 1 nt deletion, whereas the corresponding data of Lawrence et al. (10) show that 1 nt deletions constitute a minor fraction. Two major differences in the two experimental systems may account for this difference. First, we used a chemically stable synthetic AP site, whereas Lawrence et al. (9, 10) used an AP site created by treating an oligonucleotide bearing a DNA uracil with uracil glycosylase; it is possible that the synthetic AP site renders the transfected ssDNA less susceptible to spontaneous cleavage or endonucleolytic inactivation and thus may allow more translesion DNA synthesis. A second difference is that Lawrence et al. (10) used a uvrA- and umuDC-deficient E. coli strain as a host, whereas we used a uvrA+ umuDC recA strain. Finally, it should be noted that in terms of absolute numbers, 1 nt deletions are a minor event in our experiments as well, as indicated in Fig. 4A and B.

According to current understanding, the SOS mutagenesis proteins UmuD' UmuC and RecA act at the site of replication arrest to enable translesion DNA synthesis by DNA polymerase III holoenzyme. Recently, inactivation of the epsilon subunit (3'5' editing activity) of DNA polymerase III was shown to obviate the need for SOS induction for a subset of frameshift mutations induced by the carcinogen N-2-acetylaminofluorene (5). This finding has been interpreted to mean that induced SOS proteins are required only under specific circumstances to overcome the polymerase cycling forced by normal editing at lesion sites. We have noted previously that the preponderance of evidence indicates that the UVM response is mediated through an alteration of DNA replication (13). Mutation fixation at C residues was reported to be increased in mutD5 cells to the same extent as in UV-irradiated cells (11). Even though the authors of the study did not so hypothesize (11), their finding is consistent with the possibility that the UVM effect is mediated by a factor that blocks polymerase editing. The genetic requirements of the UVM response (17) suggest that the factor must be recA independent and therefore distinct from the recA-inducible hypothetical Npf factor proposed by Fuchs and Napolitano (5). The results presented here further strengthen the notion that mutagenesis observed in SOS-induced cells (i.e., cells exposed to DNA-damaging treatments) results from the operation of multiple mutagenic pathways. The knowledge that some of these pathways may not be a part of the recA- and lexA-regulated classical SOS network should facilitate a better understanding of the mechanisms underlying transient mutator responses in E. coli.