INTRODUCTION

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Stability of genetic information is a key element in the maintenance of proper cell biology and the perpetuation of species. On the other hand, evolution obviously proceeds with the help of mutations. It is thus of great interest to understand the mechanisms underlying DNA replication fidelity and its modulation. In both eukaryotic and prokaryotic organisms, DNA replication is a highly accurate process, allowing only one error among 10⁹ to 10¹⁰ incorporation events (8). However, this low error frequency of DNA replication may be enhanced either upon modification of the substrate molecule by endogenous or exogenous DNA-damaging agents, i.e., targeted mutagenesis, or through modulation of the replication fidelity in the absence of any DNA damage, i.e., untargeted mutagenesis.

In the bacterium Escherichia coli, the induction of the so-called SOS system allows the organism to cope with adverse conditions in various ways. One of the consequences of its activation is an increase in both targeted and untargeted mutagenesis (for a review, see reference 12). SOS-dependent targeted mutagenesis in E. coli relies on an increase in the frequency of DNA synthesis past DNA lesions, i.e., translesion synthesis. Although their precise mechanisms are not yet established, two distinct translesion synthesis pathways have been described to date. One relies on the activity, together with the replicative polymerase III, of the UmuDC and RecA proteins (12), whereas the other occurs independently of these accessory proteins but requires another, yet unidentified, SOS function termed Npf (17, 34, 43).

It has also been demonstrated that activation of the SOS response results in the increase of untargeted mutagenesis through the accumulation of mutations during replication of DNA that has not been exposed to any exogenous DNA-damaging agent (61). Here again, two distinct pathways can be distinguished on the basis of their specific genetic requirements. One is the so-called SOS mutator activity observed on chromosomal or episomal DNA in constitutively SOS-activated cells. This pathway requires functional recA and umuDC genes (3, 60). Strong evidence supports the notion that this mutator activity results from a transient decrease in the replication fidelity of damage-free DNA (10). The other pathway was first observed when mutations in undamaged  bacteriophages grown in UV-preirradiated E. coli cells were measured (4, 15, 32, 62). This mutagenesis, which is called  untargeted mutagenesis ( UTM), has been shown to occur independently of the umuDC function, instead requiring functional uvrABC, polA, and dinB genes (2, 7, 33, 62). As for the SOS mutator activity, some evidence supports the notion that  UTM results from a transient decrease in the replication fidelity of damage-free DNA (32).

More recently, Kim et al. (24) have shown that the expression of DinB from a low-copy-number plasmid (pYG782) is sufficient to dramatically increase the untargeted mutagenesis on F' plasmids in E. coli. This effect, subsequently termed the DinB mutator activity, has been shown to rely on the recently discovered DNA polymerase activity of DinB (DNA Pol IV [57]). Pol IV mediates template-directed DNA replication and lacks a 3'-to-5' exonuclease (proofreading) activity, and its replication mode is strictly distributive. In addition, it is prone to elongate bulged (misaligned) primer/template structures in vitro. Interestingly, at the amino acid level, DNA Pol IV shares homologies with UmuC of E. coli (also known as DNA Pol V [56]), Rev1p of Saccharomyces cerevisiae (30, 44), and DNA polymerase  of S. cerevisiae (Rad30p) and humans (also known as hRad30A or XP-V [18-20, 35-37, 51]), all of which are endowed with a DNA polymerase or a nucleotidyltransferase activity and involved in translesion DNA synthesis (11). It appears that human cells possess at least four DinB-related proteins, i.e., DNA polymerase  (hRad30A or XP-V), hRad30B, hRev1, and hDinB (14, 18, 21, 31, 36, 38, 45a).

To better characterize the DinB mutator activity in vivo, we wished to analyze the genetic requirements and mutational specificity of the DNA Pol IV mutator activity. Concerning genetic requirements, we show here that the Pol IV mutator activity acts independently of the umuDC, recA, polA, polB, and uvrA functions. Moreover, Pol IV-induced errors are correctable by the mismatch repair machinery, suggesting that they most probably represent true replication errors arising upon replication of damage-free DNA. The mutational specificity of the Pol IV mutator activity was determined by analyzing 323 cII mutants recovered from either wild-type or mismatch repair-deficient E. coli strains transformed with a low-copy-number plasmid expressing dinB (pYG782) or the control vector (pWKS30). As previously observed in another system (24), the expression of dinB greatly enhances 1 frameshift mutagenesis in this forward mutation assay. However, it also strongly promotes single nucleotide substitutions with an obvious specificity for substitutions toward G:C base pairs. Altogether, these results are discussed in terms of possible mechanisms by which DNA Pol IV mediates untargeted mutagenesis.

	MATERIALS AND METHODS

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Media, bacterial strains, and plasmids. L broth (1% Bacto Tryptone, 0.5% yeast extract, 1% NaCl [pH 7.4]) was used throughout this study. L agar contained 1.5% agar in L broth. Top agar consisted of L broth plus 0.6% agar. If necessary, ampicillin (50 µg ml¹), tetracycline (15 µg ml¹), kanamycin (20 µg ml¹), chloramphenicol (20 µg ml¹), or rifampin (100 µg ml¹) was added. All bacterial strains and plasmids used are listed in Table 1 (see the indicated references for detailed genotypes). P1vir was used for general transduction (41). Plasmid pWKS30, a pSC101 derivative containing the multiple cloning site of pBluescript II SK (59), was used for the construction of pYG782, the Pol IV-expressing vector used throughout this study. In this construction, the dinB gene is transcribed from the vector P_lac promoter (24). pMQ339 is a pACYC184 derivative containing the mutL gene (64).

Mutation assays. To measure the frequency of appearance of rifampin-resistant (Rif^r) colonies, at least two fresh transformants were independently resuspended into 3 ml of liquid L broth medium and grown overnight at 37°C with shaking. Aliquots of appropriate dilutions of these saturated cultures were then plated on agar L broth plates containing ampicillin to determine the total cell count and on plates containing rifampin (100 µg ml¹) to determine the frequency of Rif^r mutant colonies.  cII mutation frequencies were determined as follow. Overnight cultures of AB1157 derivative cells harboring the empty vector pWKS30 or the Pol IV expression vector pYG782 were concentrated by a factor of 2 in 10 mM MgSO₄. These host strains were then infected with 10⁶ PFU of untreated  phage ( cI^ts857). After 15 min of incubation at room temperature, 2 ml of L broth was added to these mixtures, and the cells were grown at 37°C with agitation until lysis occurred. After clearing the lysate by the addition of a few drops of chloroform and additional incubation at 37°C for 15 min, aliquots were centrifuged (7 min, 12,000 rpm), and 500 µl of the supernatant was saved in a fresh Eppendorf tube containing 25 µl of chloroform. Ten-microliter aliquots of the appropriate dilutions of these primary lysates were used to infect G1217 (the hfl⁺ nonselecting strain) and G1225 (the hfl mutant selecting strain) in order to determine the titer of the lysate and the  cII mutation frequency, respectively, as described by Jakubczak et al. (16). Plaques were counted after 36 to 48 h of incubation at 25°C. G1217 and G1225 strains were purchased from Epicentre Technologies as part of the MutaPlax cIISelect kit.

Mutational spectrum determination. cII mutants were selected as described above, using G1225 (hfl) and independent primary lysates resulting from infection of either AB1157 or YG2237, harboring either pWKS30 or pYG782. One plaque per independent lysate was then toothpicked and resuspended into 20 µl of 1× Pfu PCR buffer and boiled for 10 min in the PCR apparatus. The PCR mixture was then completed, and 25 cycles (15 s at 94°C, 25 s at 53°C, and 40 s at 72°C) were performed. The final composition of the PCR mixture was 1× Pfu buffer with MgSO₄, 15 pmol of each primer, 200 µM each deoxynucleoside triphosphate (TaKaRa, Shiga, Japan), and 1.25 U of cloned Pfu polymerase (Stratagene, La Jolla, Calif.). The PCR products were directly sequenced using a SequiTherm Long-Read cycle sequencing kit (Epicentre Technologies) and an ALF Red automatic sequencer (Pharmacia). The primer sequences used were the same as described by Jakubczak et al. (16) and were purchased from Pharmacia with a nonlabeled or Cy5-end-labeled oligonucleotides for sequencing purposes.

	RESULTS

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Experimental system. Originally, the  UTM phenomenon was described and analyzed through the measurement of  clear-plaque mutation frequency of undamaged bacteriophage particles grown in UV-preirradiated E. coli cells (4). Recently, Kim et al. (24) have shown that UV preirradiation of the recipient cells is not needed if DNA Pol IV is expressed from the low-copy-number plasmid pYG782. In the present study, we investigated the genetic requirements and mutational specificity of the Pol IV mutator activity by the use of two forward mutational assays, i.e., the  cII gene inactivation assay (cII assay [16]) and the rifampin resistance assay. The latter employs the rpoB gene encoding the  subunit of RNA polymerase in the chromosome of E. coli as a reporter gene for mutation and scores exclusively base substitution mutations (52). All mutagenesis experiments were carried out with cells harboring plasmid pYG782 expressing dinB or its corresponding empty vector pWKS30 as a control. No DNA-damaging treatment was applied to either the bacteria or the  particles.

Genetic requirements of the Pol IV mutator activity. In the wild-type strain, the introduction of plasmid pYG782 carrying the dinB gene induces a dramatic increase in both cII and Rif^r mutation frequencies (24- and 87-fold, respectively [Table 2]). Although some variations in the amplitude of this increase are observed, we show here that this mutator activity is independent of all gene functions tested (Table 2). The results showing the independence of the Pol IV mutator activity upon the recA, umuDC, and uvrA genes is consistent with the results obtained previously but using a target gene located on an F' plasmid (24). Results in Table 2 also demonstrate the independence of this activity on polA and polB gene functions.

Nature of the mutations induced by DNA Pol IV. Different lines of evidence have suggested that the  UTM phenomenon results from replication of nondamaged DNA (4). More recently, participation of DinB (Pol IV) in UV mutagenesis has also been investigated (24), and the results were negative. Here, using the Rif^r assay, we addressed the nature of the mutations promoted by Pol IV by analyzing the interactions between the mismatch repair pathway and Pol IV mutator activity. As depicted in Table 3, expression of the MutL protein through the introduction of plasmid pMQ339 in the cell efficiently minimized the Pol IV mutator effect in both wild-type and mutL backgrounds. On the other hand, providing additional MutL protein to a mutS strain did not affect Pol IV mutator activity, indicating that the pMQ339 effect observed in the wild-type and mutL strains indeed results from the enhancement of the mismatch repair capacities of the cell but not from some side effect of the MutL expression from pMQ339. In addition, the Pol IV mutator activity was not enhanced in a strain defective in the general nucleotide excision repair pathway mediated by the UvrABC proteins (Table 2, uvrA strain). Taken together, these findings suggest that a vast majority of the mutations caused by Pol IV are not due to the processing of cryptic DNA lesions but instead represent an amplification of true DNA replication errors.

Mutational specificity of the DNA Pol IV mutator activity. Using the positive selection system described by Jakubczak et al. (16) adapted to a bacterial study, we determined the sequence of a total of 323 independent mutants within the 294-bp-long cII gene of  phage. Briefly, E. coli host strains transformed with plasmid pYG782 or the corresponding empty vector are infected with intact  phage particles and grown until complete lysis has occurred (primary lysates). The resulting lysates are then used to infect the E. coli indicator strain that carries the hflA and hflB mutations. These infection mixtures are plated onto LB-agar plates and incubated at 25°C for 36 to 48 h. Under such conditions, only cII mutant phages are able to enter the lytic cycle and consequently form plaques on the hfl lawn. To ensure independence of such mutants, a single plaque per primary lysate is then used to amplify the entire cII gene by PCR. These PCR products are then directly sequenced. To our knowledge, this is the first extensive mutational spectrum study of untargeted mutagenesis using the cII gene as a target.

View larger version (22K): [in this window] [in a new window]   FIG. 1.   cII mutational spectra determined in E. coli AB1157 (wild-type) strain. Mutants recovered in AB1157 transformed with the pWKS30 or pYG782 vector are indicated above or below, respectively, the cII coding sequence. Base substitutions to G:C and A:T are represented in yellow and white, respectively. The G:C-directed substitutions in the sequence of 5'-GX-3', where X represents the base that is changed to G, is underlined. Blue +, +1 frameshift mutation; green triangle, 1 frameshift mutation. Other mutations are colored in orange: 2 deletion at positions 16 to 18; T insertion (iT) between positions 101 and 102; 84-bp deletion between the GTT direct repeat marked by *; +2 addition at positions 134 and 135 and -CT or -TC deletion at positions 203 to 205. Position 200 is T of a TGG sequence.

View larger version (19K): [in this window] [in a new window]   FIG. 2.   cII mutational spectra determined in E. coli YG2237 (mutS). Mutants recovered in YG2237 strain transformed with pWKS30 or pYG782 are indicated above or below, respectively, the cII coding sequence. Base substitutions to G:C and A:T represented in yellow and white, respectively. The G:C-directed substitutions in the sequence 5'-GX-3', where X represents the base that is changed to G, is underlined. Blue +, +1 frameshift mutation; green triangle, 1 frameshift mutation. Position 200 is T of a TGG sequence.

Sequence specificity of Pol IV-induced mutations. In an attempt to gain further insight into the mechanism by which Pol IV promotes mutagenesis, we looked for an eventual sequence context specificity. As described above, the patterns of mutation induction by Pol IV were very similar in both wild-type and mismatch repair-deficient strains. For the purpose of this analysis, we combined the mutational events observed in both strains. When base substitutions are considered, it appears that 70% (14/20) of the Pol IV-mediated A:T-to-G:C transitions occurred within 5'-GA-3' sequences where A is mutated to G (Fig. 1 and 2). If one looks for the same type of mutations in strains harboring the control plasmid, it appears that only 20% (2/10) occurred in such a sequence context. In the case of A:T-to-C:G transversions, 69% (11/16) occurred in 5'-GT-3' sequences where T is mutated to G. Finally, 67% (4/6) of G:C-to-C:G transversions occurred in 5'-GC-3' sequences where C is mutated to G. In summary, 69% (29/42) of the whole G:C-directed substitutions enhanced by Pol IV occurred within 5'-GX-3' sequences, where X represents A, T, or C that is mutated to G.

	DISCUSSION

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This study was conducted to further characterize the previously described DinB mutator activity which relies on the DNA polymerase activity of this protein (i.e., DNA Pol IV [57]). We show here that Pol IV does not require the functions provided by the umuDC, recA, polA, polB, or uvrA gene to promote untargeted mutagenesis (Table 2). The independence of the umuDC and recA functions is consistent with the known genetic requirements for the  UTM (2, 32, 62) and the DinB-mediated untargeted mutagenesis observed in the lacZ gene on F' plasmid (24). The independence of this mutagenic pathway of umuDC and recA distinguishes it genetically from the classical umuDC-dependent SOS mutator activity (3). On the other hand, Maenhaut-Michel and Caillet-Fauquet have shown that  UTM is not observable in uvr strains or cells that are deficient in DNA polymerase I activity (33). In addition, UV irradiation of the host cells is required for  UTM even if the SOS system is derepressed (3). In this study, however, the results clearly indicate that functional uvrA and polA genes are not necessary for the DNA Pol IV (DinB)-mediated untargeted mutagenesis (Table 2). Thus, we suggest that polymerase I activity and uvr functions play indirect rather than direct mechanistic roles in the  UTM pathway. For example, the induction of dinB might be inefficient in the polA or uvr background, and some UV-inducible but not SOS-regulated gene, such as groEL (25), may be involved in the stabilization of either DNA Pol IV itself or a not fully characterized molecular partner of the polymerase (55).

The Pol IV mutator activity is observable on either  phage or chromosomal DNA (the present work) and also on F' episomal DNA (24). It thus appears that the Pol IV mutator activity is general and does not depend on the nature of the target DNA. We used the positive selection system provided by the inactivation of the  cII gene functions to determine the specificity of the Pol IV mutator effect in a forward mutational assay (16). This selection system is very simple and appears to be quite effective since we noticed that of all the DNA of the selected plaques carried a mutation in the target gene. The ease of recovery of the target and of its sequencing also render this mutational system quite attractive. All classes of point mutations have been detected, and the recovered mutations are well distributed along the entire sequence, with perhaps the exception of the 70-nucleotide-long C-terminal part, where few base changes have been observed. Among a total of 137 base changes scored in this study, 70 represent different base substitutions, distributed over 61 different sites (in 42 out of the 98 codons). In addition, the six-G:C sequence at positions 179 to 184 clearly represent a mutational hot spot which may be useful for frameshift mutagenesis studies.

We demonstrate here that the Pol IV-induced mutations are susceptible to mismatch repair as are the mutations induced during  UTM (4), suggesting that Pol IV most probably acts at the replication fork (at least on hemimethylated DNA) and on undamaged DNA to promote true replication errors. As mentioned earlier, DNA Pol IV is a strictly distributive DNA polymerase (57). This feature implicates Pol IV in short DNA synthesis rather than in replication of the whole chromosome of E. coli or the whole  phage DNA. It is suggested that DNA Pol III holoenzyme is synthesized poorly from terminal mispairs during chromosome replication (10, 42, 48, 49). In general, purine:purine mispairs are worse substrates than purine:pyrimidine or pyrimidine:pyrimidine mispairs for extension (23, 40). Hence, we speculate that DNA Pol IV has access to the replication fork where DNA Pol III holoenzyme stalls and dissociates after it has created poorly extendable terminal mismatches. Once DNA Pol IV has access to the primer/template DNA, it carries out short DNA synthesis. This synthesis may be highly mutagenic because Pol IV lacks proofreading activity and because of its purely distributive mode of replication, as discussed below. Following dissociation of Pol IV, reassociation of the replicative Pol III holoenzyme could take place downstream from the original dissociation site and resume processive synthesis. This model could be summarized as a DNA polymerase switch (6).

The characteristics of the Pol IV-induced mutational spectrum described in this study offer clues to the mechanisms by which DNA Pol IV mediates untargeted mutagenesis (Fig. 1 and 2; Table 4). For the production of frameshifts during DNA replication, two general models have been proposed: (i) the misincorporation plus realignment model (1, 29) and (ii) the Streisinger slippage model (53, 54). The former can occur favorably if the misincorporated base is complementary to the next, i.e., 5', template base; the misalignment allowing further synthesis to proceed from a correctly paired 3' terminus. This type of mechanism is generally associated with frameshifts occurring at nonreiterated sequences in specific sequence contexts and with poorly processive DNA polymerases (1, 27). In contrast, frameshifts thought to be generated through the Streisinger slippage model are associated with runs of identical bases, and their probability of occurrence increases with the length of the run (28, 54). Slippage errors in runs are generated during processive DNA replication (13).

In this study, 1 frameshift mutations are the predominant mutations observed in the cII gene when DNA Pol IV is expressed (Table 4; Fig. 1 and 2). Among them, the six-G:C sequence at positions 179 to 184 is the most sensitive site for Pol IV mutator activity. Such hot spots in runs are generally considered an indication for the Streisinger slippage model. Thus, it seems that the frameshifts produced within the six-G:C hot spot result from direct slippage errors by DNA Pol IV. The slippage errors may be enhanced when Pol IV interacts with  subunit of Pol III holoenzyme (55). Besides run sequences, frameshift mutagenesis was efficiently promoted by Pol IV in short runs or even in nonrun sequences. In addition, the mutations in short runs or nonrun sequences occur predominantly in a specific sequence context, i.e., sequences harboring a 5' G next to the mutated base. Thus, it seems possible that some of Pol IV-mediated frameshift mutagenesis occur by the misincorporation-realignment mechanism. Unlike processive DNA synthesis, only a single nucleotide is incorporated during each encounter of DNA Pol IV with the template/primer DNA (57). This feature may increase the realignment probability of the DNA strands, thereby promoting the formation of a 1-bp looped-out structure (Fig. 3, step 1). Our recent in vitro studies have highlighted the propensity of DNA Pol IV to extend, within a three-G:C context, a preexisting terminal mismatch through the formation of a 1-bp loop in the template strand (57).

View larger version (15K): [in this window] [in a new window]   FIG. 3.   Model for Pol IV-induced mutagenesis leading to a 1-bp deletion and single-nucleotide substitution. The primer strand of a replication intermediate is extended by DNA Pol III holoenzyme (Pol III HE). The model assumes that Pol III HE dissociates from DNA when it creates a 3'-terminal C:C mismatch. If the matched primer terminus having an extrahelical C is efficiently extended by Pol IV, a 1-bp deletion will be generated (step 1). DNA Pol IV may also catalyze the direct extension from the mismatched primer terminus (step 2), thereby generating a G:C-to-C:G transversion mutation. The DNA sequences are those at positions 208 to 215 of the cII gene of  phage, where the two types of mutations are observed. The underlined base is position 211 of the cII gene of  phage shown in Fig. 1 and 2.

Base substitutions account for about one-third of the total mutations induced by Pol IV. Intriguingly, upon expression of DNA Pol IV in the wild-type strain, base changes toward G:C (i.e., A:T to G:C, T:A to G:C, and C:G to G:C) are enhanced 121- to 170-fold (18- to 147-fold in the mutS strain [Table 4]) and largely outnumber other base substitutions. Assuming the aforementioned DNA polymerase switch model, this bias may be explained by a strong difficulty for Pol III holoenzyme to elongate particular terminal mismatches. This difficulty will result in an increased probability for Pol III to dissociate, thus giving a chance to the proofreading deficient Pol IV to elongate the mismatch (Fig. 3, step 2). We noticed that about 70% of these base substitutions occurred within a 5'-GX-3' sequence context where X represents the mutated base. The 5'-proximal neighbor effect in the template strand is described for base replacements by other DNA polymerases (9, 26, 28, 29). Thus, the sequence context may suggest a particular ease for DNA Pol IV to extend a mismatched primer terminus by the incorporation of a C residue. Interestingly, the mutations observed at positions 210 and 211 of the cII gene are a 1-bp deletion, i.e., 5'-GC-3' to 5'-G-3', and a base change, i.e., 5'-GC-3' to 5'-GG-3', in both wild-type and mutS backgrounds. Competition may occur between the pathway leading to a 1-bp deletion (Fig. 3, step 1) and that leading to a base substitution (Fig. 3, step 2) at the same site. Nevertheless, 5'-GX-3' sequence (the lower sequence in Fig. 3) might act as a primer strand, and the opposite strand might be the template. In that case, an A insertion opposite G at position 210 would allow ready slippage on T209, causing a 1 frameshift, and G misinsertion opposite G211 might yield a G:C-to-C:G transversion. It is reported that 5' base in the primer strand (G212 in this case) affects the misinsertion rate (at position 211) through stacking interactions (39, 47). Thus, more focused mutational and biochemical studies are needed to elucidate the role of this sequence context.

In a previous paper, Kim et al. reported that base changes as well as frameshifts are induced by DinB (DNA Pol IV) expression in the lacZ gene on F' plasmid (24). However, the extent of induction of these base changes observed in the F' system is much lower than the extent of induction of base changes in the cII gene in the present study, although the amplitudes of frameshift induction in the run of six G:C are comparable between the lacZ and cII assays. Notably, the G:C-directed base substitutions are poorly detected in the lacZ reversion assay: the frequency of A:T to G:C, T:A to G:C, and C:G to G:C changes are enhanced 4, 17, and 4 times, respectively, by Pol IV expression. Since the lacZ assay is a reversion assay, only a specific type of mutation in a specific sequence context can revert the phenotype from LacZ to LacZ⁺. E. coli CC101 detects T:A-to-G:C base changes in the sequence 5'-AATTAG-3' where the underlined T is the target base. Similarly, strains CC103 and CC106 detect C:G to G:C and A:T to G:C within the sequences 5'-AATCAG-3' and 5'-AATAAG-3', respectively. None of these sequences include 5'-GX-3', which is the sequence favored for base substitutions generated by DNA Pol IV. Thus, the G:C-directed base changes could be induced in the lacZ gene by Pol IV but are poorly detectable in the lacZ reversion assay because of the sequence context. In this respect, a forward mutation assay such as that using the cII gene in this study reflects more genuinely the mutation spectra generated by DNA Pol IV than the reversion assay.

This study represents an in vivo analysis of the mutational specificity of DNA Pol IV (DinB), which belongs to the ubiquitous family of the very recently discovered novel DNA polymerases involved in mutagenesis (reviewed in reference 11). The data obtained should help to direct future studies aimed at better characterizing the biochemical specificities of this novel DNA polymerase and the molecular basis of mutagenesis.