Although very little replication past a T-T cis-syn cyclobutane dimer normally takes place in Escherichia coliin the absence of DNA polymerase V (Pol V), we previously observed as much as half of the wild-type bypass frequency in Pol V-deficient (⌬umuDC) strains if the 3 to 5 exonuclease proofreading activity of the Pol III subunit was also disabled by mutD5. This observation might be explained in at least two ways. In the absence of Pol V, wild-type Pol III might bind preferentially to the blocked primer terminus but be incapable of bypass, whereas the proofreading-deficient enzyme might dissociate more readily, providing access to bypass polymerases. Alternatively, even though wild-type Pol III is generally regarded as being incapable of lesion bypass, proofreading-impaired Pol III might itself perform this function. We have investigated this issue by examining dimer bypass frequencies in ⌬umuDC mutD5 strains that were also deficient for Pol I, Pol II, and Pol IV, both singly and in all combinations. Dimer bypass frequencies were not decreased in any of these strains and indeed in some were increased to levels approaching those found in strains containing Pol V. Efficient dimer bypass was, however, entirely dependent on the proofreading deficiency imparted by mutD5, indicating the surprising conclusion that bypass was probably performed by the mutD5 Pol III enzyme itself. This mutant polymerase does not replicate past the much more distorted T-T (6-4) photoadduct, however, suggesting that it may only replicate past lesions, like the T-T dimer, that form base pairs normally.The discovery that Escherichia coli possesses two new DNA polymerases, Pol IV and Pol V (26,32,38), in addition to the three that had been previously identified (18) raises the questions of why E. coli needs five DNA polymerases and what the cellular functions of each might be. At present, it is believed that DNA Pol III is responsible mainly for the replication of undamaged templates, that Pol I participates in filling gaps between Okazaki fragments and those generated during nucleotide excision repair (7,18), and that Pol II participates in replication restart after UV irradiation (25). In addition, Pol II, along with Pol IV and Pol V, has been implicated in translesion replication (TR) (22, 31). However, in many cases, such roles appear to be polymerase and lesion specific. For example, previous observations (31, 36) have suggested that TR past a site-specific cis-syn cyclobutane thymine-thymine dimer in excision-defective E. coli strains is almost entirely dependent on the activity of DNA polymerase V (Pol V), encoded by the umuDC operon. Curiously, however, we also observed that efficient TR past cis-syn cyclobutane dimers occurs in SOSinduced ⌬umuDC cells lacking Pol V, if the 3Ј to 5Ј exonuclease proofreading activity of the DNA polymerase III ε subunit is inactivated by the mutD5 mutation (36). Indeed, 26% TR occurred in UV-irradiated uvrA6 ⌬umuDC mutD5 cells, about half the frequency found in similarly treated isogenic uvrA6 umuDC ϩ...

Translesion replication (TR) past a cyclobutane pyrimidine dimer in Escherichia coli normally requires the UmuD'2C complex, RecA protein, and DNA polymerase III holoenzyme (pol III). However, we find that efficient TR can occur in the absence of the Umu proteins if the 3'-5' exonuclease proofreading activity of the pol III epsilon-subunit also is disabled. TR was measured in isogenic uvrA6 DeltaumuDC strains carrying the dominant negative dnaQ allele, mutD5, or DeltadnaQ spq-2 mutations by transfecting them with single-stranded M13-based vectors containing a specifically located cis-syn T-T dimer. As expected, little TR was observed in the DeltaumuDC dnaQ+ strain. Surprisingly, 26% TR occurred in UV-irradiated DeltaumuDC mutD5 cells, one-half the frequency found in a uvrA6 umuDC+mutD5 strain. lexA3 (Ind-) derivatives of the strains showed that this TR was contingent on two inducible functions, one LexA-dependent, responsible for approximately 70% of the TR, and another LexA-independent, responsible for the remaining approximately 30%. Curiously, the DeltaumuDC DeltadnaQ spq-2 strain exhibited only the LexA-independent level of TR. The cause of this result appears to be the spq-2 allele, a dnaE mutation required for viability in DeltadnaQ strains, since introduction of spq-2 into the DeltaumuDC mutD5 strain also reduces the frequency of TR to the LexA-independent level. The molecular mechanism responsible for the LexA-independent TR is unknown but may be related to the UVM phenomenon [Palejwala, V. A., Wang, G. E., Murphy, H. S. & Humayun, M. Z. (1995) J. Bacteriol. 177, 6041-6048]. LexA-dependent TR does not result from the induction of pol II, since TR in the DeltaumuDC mutD5 strain is unchanged by introduction of a DeltapolB mutation.

In wild-type Escherichia coli, translesion replication is largely dependent upon the UmuD 2 C complex (DNA polymerase V [polV]) or its plasmid-encoded homologs, such as MucA 2 B. Interestingly, both the efficiency of translesion replication of a T-T cis-syn dimer and the spectra of mutations observed are different in Umu-and Muc-expressing strains. We have investigated whether the polIII core is responsible for these differences by measuring the frequency of dimer bypass, the error rate of bypass, and the resulting mutation spectrum in mutants carrying a deletion of dnaQ ( subunit) or holE ( subunit) or carrying the dnaQ allele mutD5, which is deficient in proofreading but is competent in the structural function of , or the dnaE antimutator allele spq-2. The chromosomal copy of the umuDC operon was deleted in each strain, and the UmuDC, UmuDC, MucAB, or MucAB proteins were expressed from a low-copy-number plasmid. With only few exceptions, we found that the characteristically different mutation spectra resulting from Umu-and Muc-mediated bypass are maintained in all of the strains investigated, indicating that differences in the activity or structure of the polIII core are not responsible for the observed phenotype. We also demonstrate that the MucA 2 B complex is more efficient in promoting translesion replication than the UmuD 2 C proteins and show that, contrary to expectation, the T-T dimer is bypassed more accurately by MucA 2 B than by UmuD 2 C. These results are consistent with the view that in a wild-type cell, the polV-like enzymes are responsible for the spectra of mutations generated during translesion replication and that polIII may simply be required to fix the misincorporations as mutations by completing chromosomal replication. Our observations also show that the mutagenic properties of a lesion can depend strongly on the particular enzyme employed in bypass.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 UmuDЈ 2 C 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 3Ј-5Ј exonucleolytic proofread...