Escherichia coliDNA Polymerase III Can Replicate Efficiently past a T-Tcis-synCyclobutane Dimer if DNA Polymerase V and the 3′ to 5′ Exonuclease Proofreading Function Encoded bydnaQAre Inactivated

Abstract

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...

Recruitment of host functions suggests a repair pathway for late steps in group II intron retrohoming

“…Which of the three repair polymerases replaces the displaced Pol III in a complex depends on timing, enzyme availability, and the type of DNA lesion (Crowley and Courcelle 2002;Goodman 2002;Wagner et al 2002). Our results indicate that in the Pol V mutant, retrohoming increases by 50% (Table 3), which may be consistent with the observation that Pol III can replicate efficiently past some DNA lesions when Pol V is inactivated (Borden et al 2002). Although the lack of Pol II or Pol IV can reduce retrohoming by two-thirds, the Pol II-Pol IV double mutant does not have an additive effect.…”

Essential roles for imuA ′- and imuB -encoded accessory factors in DnaE2-dependent mutagenesis in Mycobacterium tuberculosis

“…S3), which suggests the potential for other intermolecular interactions. In E. coli, replicative polymerase fidelity is enhanced by the association of the α-subunit with the dnaQ-encoded proofreading exonuclease, and disruptions to proofreading activity enable DNA polymerase III-mediated lesion bypass in the absence of DNA polymerases IV and V (31,32). In combination, these observations suggest that differential interactions with DnaQ-like proteins might determine DnaE subunit function in Mtb.…”

Involvement of DnaE, the Second Replicative DNA Polymerase from Bacillus subtilis, in DNA Mutagenesis

“…This holds true for damage-containing templates as shown with the T7 pol and the E. coli pol I, pol II, and pol III enzymes (67)(68)(69)(70). Thus, if DnaE has a proofreader, it is likely that the DnaE-proofreader complex exhibits a lower bypass capacity than described here for the purified DnaE protein.…”