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SummaryAlthough best characterized for their ability to traverse a variety of DNA lesions, Y-family DNA polymerases can also give rise to elevated spontaneous mutation rates if they are allowed to replicate undamaged DNA. One such enzyme that promotes high levels of spontaneous mutagenesis in Escherichia coli is polV R391, a polV-like Y-family polymerase encoded by rumAЈB from the IncJ conjugative transposon R391. When expressed in a DumuDC lexA(Def) recA730 strain, polVR391 promotes higher levels of spontaneous mutagenesis than the related MucAЈB (polR1) or UmuDЈC (polV) polymerases respectively. Analysis of the spectrum of polVR391-dependent mutations in rpoB revealed a unique genetic fingerprint that is typified by an increase in C:G,A:T and A:T,T:A transversions at certain mutagenic hot spots. Biochemical characterization of polVR391 highlights the exceptional ability of the enzyme to misincorporate T opposite C and T in sequence contexts corresponding to mutagenic hot spots. Purified polVR391 can also bypass a T-T pyrimidine dimer efficiently and displays greater accuracy opposite the 3ЈT of the dimer than opposite an undamaged T. Our study therefore provides evidence for the molecular basis for the enhanced spontaneous mutator activity of RumAЈB, as well as explains its ability to promote efficient and accurate bypass of T-T pyrimidine dimers in vivo.

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.