INTRODUCTION

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It has been inferred, on the basis of a variety of genetic evidence obtained over the last 20 years, that translesion replication and DNA damage-induced mutagenesis (SOS mutagenesis) in Escherichia coli are dependent on the activity of DNA polymerase III (polIII) holoenzyme or a modified form of it (4, 5, 14, 26, 27; reviewed in reference 10). Although polIII is usually unable to perform lesion bypass by itself, it can do so with the aid of what were previously thought to be accessory factors, the chromosomally encoded UmuDC proteins or their plasmid-encoded homologs, such as MucAB (16, 22, 30). However, the recent discovery that the UmuD'₂C protein complex, now called polV, itself possesses intrinsic DNA polymerase activity (24, 25, 32, 33) raises the question of which enzyme is responsible for mutagenesis. Is polV solely responsible, or do some or all of the polIII subunits also influence SOS mutagenesis in vivo? In vitro studies show that polV is capable of both nucleotide incorporation opposite an abasic site and extension from this terminus in the absence of the polIII core (composed of the catalytic  subunit, the 3'-5' exonucleolytic proofreading  subunit, and the  subunit, whose function is not yet known). The addition of a low level of polIII to the in vitro reaction mixture nevertheless stimulates elongation by some as yet undefined mechanism (33), raising the possibility that one or more of the polIII core subunits might influence efficiency of lesion bypass in vivo.

Although most attention has been given to translesion replication employing the Umu proteins, many homologs encoded on naturally occurring R plasmids (30) are capable of providing a substitute function, which presumably is also a DNA polymerase activity. These include the mucAB operon from the incN plasmid R46 and its better-characterized deletion derivative pKM101 (22) and the rumAB operon from the incJ plasmid R391 (18). Although these homologs are clearly related structurally and belong to the same branch of the UmuC/DinB/Rev1/Rad30 superfamily of DNA polymerases (21), they are not entirely equivalent with respect to their mutagenesis-promoting properties (1, 18, 35). A particularly clear example of this lack of equivalence is the difference in the predominant type of mutation that occurs during Umu- or Muc-facilitated replication past a site-specific T-T cis-syn cyclobutane dimer. Earlier studies (2, 19) using SMH10, a uvrA6 derivative of AB1157 which contains a normal chromosomal copy of the umuDC operon, showed that almost all of the errors induced were 3' TA and 3' TC mutations and that there were about fivefold (130:28) more transversions than transitions. A similar result was found in experiments using RW82, another uvrA6 derivative of AB1157 in which the chromosomal copy of the umuDC operon is deleted (37) and in which the umu genes were carried on a low-copy-number plasmid (31). However, the ratio of transitions to transversions was reversed when Muc proteins were substituted and the predominant mutation was 3' TC. The MucA'₂B and UmuD'₂C complexes also differed with respect to bypass efficiency; the MucAB proteins promoted bypass more efficiently than their Umu counterparts, in keeping with their known ability to enhance DNA damage-induced mutation frequencies above those usually seen in strains expressing the umuDC operon (1, 18, 35). Even so, the error rate of MucA'₂B-assisted bypass did not appear to be higher; indeed, if anything, it was lower.

A variety of mechanisms might cause differences in the mutagenic properties of a T-T dimer when Muc rather than Umu proteins are employed. They might arise from different inherent error-making properties of the DNA polymerase that inserts nucleotides opposite the lesion. However, if this is the case, such a phenomenon would be highly unusual because the same predominant type of mutation appears to be determined by the structure of the lesion rather than the enzyme; even though the error rate may vary, the same major type of mutation is normally induced by a given lesion, even when introduced into very different enzymatic environments, such as those found within yeast and E. coli cells (2, 3, 11, 12, 19, 20). Alternatively, if the polIII holoenzyme is involved in translesion replication in vivo, it might, according to whether the Umu or Muc proteins were involved, elongate differentially from the T · T and T · G mispairs which are responsible for the mutations observed. Moreover, these mismatches might be subject to differential proofreading by the 3'-5' exonuclease activity of the polIII  subunit encoded by dnaQ.

The aim of the work described in this report was, therefore, to reassess the respective roles of the polIII core and polV or its functional homolog MucA'₂B in SOS mutagenesis in vivo. To this end, we have examined the frequency of translesion replication past a single T-T cis-syn cyclobutane dimer, together with the error frequency of bypass and the mutation spectra, in various E. coli strains containing mutations or deletions of each of the polIII core subunits (, dnaE; , dnaQ; , holE) in the presence of the E. coli UmuDC proteins or their highly active plasmid-encoded homologs MucAB. We found that the mutation spectra characteristic of Umu and Muc proteins are maintained in almost all of the strains and under almost all of the conditions investigated, indicating that differences in the activity or structure of the polIII core are not responsible for the observed phenotype. We also found that the MucA'₂B complex is more efficient at promoting translesion replication than the UmuD'₂C proteins and that, contrary to expectation, bypass of the T-T dimer dependent on MucA'₂B is more accurate than that mediated by UmuD'₂C. Such findings show that a lesion's mutagenic parameters can be greatly influenced by the properties of the polymerase employed in translesion replication.

	MATERIALS AND METHODS

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Bacterial strains and plasmids. The bacterial strains and plasmids used in this study are listed in Table 1. The strains are all isogenic with uvrA6 mutant strain TK603 (17). Most have been described before, and details of their construction are given in reference 31. The exception is DV01, which was made by transducing the holE202::cat allele from RM4193 into EC8 and selecting for chloramphenicol resistance (29). The construction of plasmids expressing the UmuDC or MucAB proteins or their mutagenically active counterparts UmuD'C and MucA'B is described in reference 31.

Preparation of vectors with a site-specific T-T cis-syn cyclobutane dimer. Single-stranded vectors based on the hybrid phage M13mp7L2 (2) and carrying a specifically located T-T cis-syn cyclobutane dimer were constructed as described previously (2, 3). In this method, viral DNA from M13mp7L2 is linearized by digestion with EcoRI, which cuts within the small hairpin region, and the linear DNA is recircularized by annealing with a 51-mer scaffold oligomer. The ends of the scaffold are complementary to the terminal 20 nucleotides at each of the ends of the linearized vector, which are therefore separated by 11 nucleotides. An 11-mer with a unique T-T dimer is then ligated efficiently into this gap, and the scaffold is removed by heat denaturation in the presence of a large excess of an antisense 51-mer which has a sequence complementary to that of the scaffold. The control vector is constructed from equal aliquots of the recircularized material in identical reactions carried out with the unmodified 11-mer. Under the conditions used, ligation efficiencies are >90% and equal for the control and modified materials. Molecules containing the dimer are produced by exposing 50 µg of the oligomer 5' GCAAGTTGGAG 3' in 100 µl of anoxic aqueous acetophenone (2 × 10² M) to ~250 kJ of filtered sunlamp radiation (>315 nm) per m². The desired species is purified by high-performance liquid chromatography and repeatedly repurified to achieve >99.5% purity.

Other methods. Transfection procedures and analysis of the replicated vector sequence were done as previously described (2, 3). Where indicated, cells were irradiated with 4 J of 254-nm UV per m² immediately before being made competent with CaCl₂, to induce the SOS regulon. Five hundred microliters of irradiated or unirradiated competent cells was transfected with 5 ng of control or lesion-containing construct DNA, and the resulting plaques were counted. Only 1.5 ng of DNA was used with strain DV02 because it was highly transformable. Conversely, 7.5 ng of DNA was used with the poorly transformable DV05 strain. The number of plaques from the dimer-containing construct normalized to the control numbers was used to estimate the frequency of translesion replication. Replication events at the dimer target site were determined by hybridization and sequence analysis (2, 3).

	RESULTS

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Influence of mutations in each of the three subunits of the polIII core on Umu- and Muc-dependent spectra. The difference between the mutation spectra of cells expressing the Umu and Muc complexes is well illustrated by the data from RW82 (Table 2). When this umuDC strain contained an episomal copy of either umuDC or umuD'C, 3' TA transversions were always severalfold more abundant than the transition, whether the cells were UV irradiated or not. In contrast, when the strain expressed either the MucAB or MucA'B proteins, the reverse was true in all cases. Results from strains carrying an episomal umuDC operon are closely similar to those obtained with the intact chromosomal genes, even though there are probably three to five plasmids per cell, and thus the cells are likely to contain more Umu protein. The ratios of transitions to transversions were not significantly heterogeneous (²_[3] = 3.37; P = 0.5 to 0.1) in UV-irradiated cells of RW82 with either umuDC or umuD'C on the plasmid, compared to those from SOS-induced cells of the isogenic parent strain TK603 and its mutD5 derivative DV12, both of which carry intact chromosomal operons. Results obtained with unirradiated TK603 and DV12 cells appear to be different, but this is probably misleading because of the very low bypass frequencies. When bypass frequencies are appreciable, replication of virtually all vector molecules entails dimer bypass. However, when bypass frequencies are very low, as in unirradiated TK603 and DV12 cells, a significant fraction of replicated vectors can result from the small trace of vectors lacking the dimer. The purity of the dimer-containing oligonucleotides used to construct the vector was >99.5%, but the remaining <0.5%, most of which is probably dimer-free oligomer, becomes significant when bypass frequencies in lesion-containing vectors are only a few percent.

View this table: [in this window] [in a new window]   TABLE 2.   Number of replicated vector molecules with a normal or mutant sequence at the T-T dimer site, ratio of 3' TA to 3' TC mutations, and bypass error rate in uvrA6 strains expressing (where indicated) UmuDC or MucAB proteins or their mutagenically active counterparts UmuD'C or Muc A'B from low-copy-number plasmids

Bypass accuracy is greater with Muc than with Umu proteins. In addition to the ratios of the different types of mutations, another important parameter of mutagenesis is the overall error rate of translesion replication. Because the Muc proteins vigorously promote mutagenesis (18, 35), it might be expected that dimer bypass replication facilitated by these proteins would be less accurate than bypass using the Umu gene products. Previous results failed to detect the expected increase in error rate, and indeed, if anything, the error rate seemed lower, although the data were inadequate to establish this point. The present results (Table 2), however, clearly indicate that this is the case. Using the data from RW82, DV02, DV03, and DV05, the overall error rate in strains expressing the Muc proteins is only a little over half of that in strains containing the Umu gene products (3.4% in Muc strains, 6.3% in Umu strains). For the most part, the difference is seen consistently with all of the strains and conditions, although the results from strain DV02 with the mucA'B plasmid are, for unknown reasons, anomalous. Nevertheless, Muc- and Umu-dependent error rates are, on average, very significantly different in both UV-irradiated (²_[1] = 82.4; P 0.001) and unirradiated (²_[1] = 20.6; P 0.001) cells. Again, the results from the Umu-containing strains seem quite typical of those from strains with a normal chromosomal copy of umuDC. The average error rate in the plasmid-containing UV-irradiated strains is a little higher than the average value from TK603 and DV12 (Table 2), in which umuDC is chromosomally expressed, but the difference is short of significance (²_[1] = 3.73; P = 0.1 to 0.05). Moreover, an error rate of 6.3% in the umu plasmid-containing strains was identical to that previously observed in a large set of data from a strain with genomic umuDC (19). These results indicate not only that the error rate is independent of the location of the umuDC operon but also that it is independent of the level of Umu or Muc proteins and the amount of bypass.

Effect of proofreading or the  subunit on lesion bypass frequency in cells containing Umu or Muc proteins. A variety of evidence suggests that the 3'-5' exonucleolytic proofreading activity of the polIII  subunit plays little or no role in induced mutagenesis and lesion bypass (8, 36, 38), but the high spontaneous mutability of strains lacking this activity has made it difficult to obtain decisive results. Slater and Maurer (28) have clearly shown, using a dnaQ spq-2 strain, that the dependence on MucAB for efficient replication of UV-irradiated X174 phage DNA cannot be relieved by absence of the  subunit. Their experiments did not differentiate between the structural and proofreading roles of , however, or examine possible effects of the spq-2 mutation, a dnaE allele also present in their strain, and the system used was relatively insensitive to small changes in bypass frequency. Experiments using vectors uniformly carrying a single lesion have the advantage that they provide direct measures of bypass frequency and are unaffected by high spontaneous mutation rates.

View this table: [in this window] [in a new window]   TABLE 3.   Effects of Umu-like proteins on translesion replication in umuDC uvrA6 strains carrying dnaQ+, dnaQ, spq-2, or mutD5 alleles

	DISCUSSION

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The chief aim of this work has been to investigate whether the E. coli DNA polIII core plays a role in vivo in determining the differences observed between the mutagenic properties of polV (UmuD'₂C) and the plasmid-encoded homolog of polV (MucA'₂B) during replication past a T-T dimer. Results presented in this and an earlier paper (31) show that Muc-dependent bypass differs from that mediated by polV in three respects. First, substituting Muc for Umu proteins drastically alters the ratio of the two major classes of mutation induced by the dimer. In strains with a normal chromosomally located umuDC operon, almost all mutations occur at the site of the 3' thymine and about 80% are TA transversions, with the remainder being TC transitions (19). The same mutations and ratio were also found in strains in which the chromosomal operon was deleted and the Umu proteins were produced from genes carried on a low-copy-number plasmid (Table 2 and reference 31). However, substitution of mucAB for umuDC on the plasmid reversed the ratio of the mutant classes, with the transition now being the major class of mutations. Second, T-T dimer bypass with Muc rather than Umu proteins is nearly twice as accurate; the average error rate was 3.4%, compared to 6.3% in Umu-containing strains (Table 2). Third, the Muc proteins are generally more efficient at promoting bypass and result in a higher proportion of the vector molecules being fully replicated (Table 3; reference 31). Investigation of the mechanisms responsible for these differences has the potential of increasing our understanding of the enzymology of bypass in vivo.

Based upon earlier genetic work (4, 5, 14, 26, 27; reviewed in reference 10), we initially hypothesized that such differences would result from a modification of polIII. However, given the finding that UmuD'₂C is an error-prone polymerase, polV, and that MucA'₂B almost certainly possesses a similar activity, our observations are most easily explained by the fact the characteristic mutation spectra of Umu- and Muc-dependent dimer bypass simply reflect the inherent properties of DNA polV and its MucA'₂B counterpart. An enzyme-dependent difference in error propensity of this magnitude is unusual because previous studies suggested that the mutation spectrum is largely determined by the structure of the particular lesion concerned. Even though the overall error frequency might vary, the same predominant type of mutation was induced by a given lesion when introduced into the very different enzymatic environments found within yeast and E. coli cells. Thus, a 3' TC mutation is the major type of event induced by a T-T pyrimidine (6-4) pyrimidinone adduct in both species (12, 20) and in each of them, a 5' T-to-A mutation is the predominant event induced by a T-T trans-syn cyclobutane dimer (3, 11). Whether the mutations induced by a cis-syn dimer are similar in the two species is hard to determine, since almost no mutations are induced by this photoproduct in yeast (11). Thus, it is unusual if, as seems likely, the differences in mutation spectrum are caused by the inherent error propensities of polV and its MucA'₂B counterpart. Structural studies of the two polymerases cocrystallized with primed T-T dimer-containing templates are likely to be useful in finding molecular mechanisms for such a difference.

The data in Table 2 clearly demonstrate that the 3'-5' exonuclease proofreading function of the  subunit of the polIII holoenzyme has no influence on the mutation spectrum in Umu- and Muc-containing cells. Our present data are also incompatible with models that explain the difference in mutation spectra based on differential extension from T · T and T · G mismatches, because the difference between the mutation patterns induced in the presence of Umu or Muc proteins was independent of the proportion of vector molecules in which translesion replication occurred. Assuming a frequency of mispair formation independent of Umu or Muc, elongation preference will have the greatest capacity to change the ratio of the mutations recovered when few bypass events occur. It will have a decreasing effect as the frequency of bypass increases and will not occur at all if 100% of the molecules are fully replicated. Contrary to this expectation, the ratios characteristic of Umu- and Muc-expressing cells were maintained even when translesion replication occurred in 80% of the vector molecules in Umu-containing cells and in 100% of the vector molecules in Muc-containing cells (Tables 2 and 3).

The finding that bypass using the Muc proteins is nearly twice as accurate as bypass with polV is surprising in view of the known capability of the muc operon to promote higher levels of DNA damage-induced mutagenesis than those found with umu. Although it is possible that this result is restricted to dimers, with higher error rates being found with other lesions, the increased ability of the Muc proteins to elevate induced mutation frequencies more probably resides in their superior capacity to promote translesion replication. Part of this superior capacity probably reflects a greater susceptibility of the MucA protein to posttranslational processing by activated RecA (15). However, a recent study (33) suggests that much lower levels of MucA'₂B than polV are sufficient for high levels of mutagenesis, indicating that MucA'₂B is likely to possess inherently greater activity for promoting nucleotide insertion and extension opposite any lesion.

Reassessment of the role of the polIII core in SOS mutagenesis. Our observation that polV and MucA'₂B exhibit substantially different mutation spectra and a twofold difference in error rate clearly indicates that these enzymes play an important role at the site of the 3' thymine in the dimer, the first encountered by polymerase, and this places limitations on a possible role for the polIII core in translesion replication. Moreover, the mutagenic properties are essentially unchanged over a wide range of expression levels of polV and MucA'₂B (for example, UmuDC without SOS induction and UmuD'C or MucA'B with SOS induction), conditions that should substantially change the levels of the polV-like enzymes relative to that of polIII. If both polIII and the polV-like enzymes were active in bypass, the mutagenic properties would be expected to vary, but this is not observed. Moreover, the absence of any influence of proofreading on the mutagenic properties of the dimer and earlier evidence indicating the absence of an effect of proofreading on UV-induced mutagenesis (38) argue against extension by polIII. To date, no plausible molecular mechanism has been suggested for the hypothesized suppression of the  subunit's proofreading activity during translesion replication and its restoration immediately beyond the lesion. In contrast, replication by polV or its counterparts (which appear to lack proofreading activity) is a much more persuasive explanation. Thus, we believe that in a wild-type cell, polV and its plasmid-encoded homologs perform both the misincorporation and extension steps in translesion replication. Indeed, recent biochemical analysis of polV supports such an assumption (32). As polV is a distributive enzyme (32, 32a, 33), it is almost certainly replaced by the more processive enzyme polIII once the kinetic block to replication has been alleviated and allows polIII to fix the polV-dependent misincorporated base as a mutation.