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. 2008 Mar;190(5):1730-42.
doi: 10.1128/JB.01463-07. Epub 2007 Dec 21.

Role of accessory DNA polymerases in DNA replication in Escherichia coli: analysis of the dnaX36 mutator mutant

Damian Gawel  1 , Phuong T Pham, Iwona J Fijalkowska, Piotr Jonczyk, Roel M Schaaper
Affiliations

Role of accessory DNA polymerases in DNA replication in Escherichia coli: analysis of the dnaX36 mutator mutant

Damian Gawel et al. J Bacteriol. 2008 Mar.

Abstract

The dnaX36(TS) mutant of Escherichia coli confers a distinct mutator phenotype characterized by enhancement of transversion base substitutions and certain (-1) frameshift mutations. Here, we have further investigated the possible mechanism(s) underlying this mutator effect, focusing in particular on the role of the various E. coli DNA polymerases. The dnaX gene encodes the tau subunit of DNA polymerase III (Pol III) holoenzyme, the enzyme responsible for replication of the bacterial chromosome. The dnaX36 defect resides in the C-terminal domain V of tau, essential for interaction of tau with the alpha (polymerase) subunit, suggesting that the mutator phenotype is caused by an impaired or altered alpha-tau interaction. We previously proposed that the mutator activity results from aberrant processing of terminal mismatches created by Pol III insertion errors. The present results, including lack of interaction of dnaX36 with mutM, mutY, and recA defects, support our assumption that dnaX36-mediated mutations originate as errors of replication rather than DNA damage-related events. Second, an important role is described for DNA Pol II and Pol IV in preventing and producing, respectively, the mutations. In the system used, a high fraction of the mutations is dependent on the action of Pol IV in a (dinB) gene dosage-dependent manner. However, an even larger but opposing role is deduced for Pol II, revealing Pol II to be a major editor of Pol III mediated replication errors. Overall, the results provide insight into the interplay of the various DNA polymerases, and of tau subunit, in securing a high fidelity of replication.

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Figures

FIG. 1.
FIG. 1.
Effect of Pol IV (dinB) and Pol V (umuDC) on the dnaX36 mutator effect. All strains used were mismatch repair deficient (mutL). Mutant frequencies were determined as described in the Materials and Methods. (A) Mutant frequencies for lac G·C→T·A transversions; (B) mutant frequencies for trpE9777 reversion (−1 frameshift). The figure shows the results of one representative experiment. These experiments were performed multiple times, which yielded similar results. The dnaX+ strains used were NR13153, NR13157, NR13155, and NR13159. The dnaX36 strains were NR13256, NR13272, NR13258, and NR13274. The dnaX+ ΔumuDC strains were NR13312, NR13316, NR13314, and NR13318. The dnaX36 ΔumuDC strains were NR13276, NR13292, NR13278, and NR13296. The status of the dinB gene, chromosomal and/or episomal, in these strains is indicated in the boxes below the graph.
FIG. 2.
FIG. 2.
Effect of Pol II (ΔpolB and polBex1) and Pol IV (dinB) on the dnaX36 mutator activity. The dinB strains lack both chromosomal and episomal gene copies. All strains are also mismatch repair deficient (mutL). Mutant frequencies were determined as described in Materials and Methods. In this series of experiments, the background mutant frequency for the lac G·C→T·A was higher than in previous experiments (Table 2 and Fig. 1). The present experiments were performed at a later time and under slightly different conditions. Nevertheless, within this series of experiments the results were highly consistent and reproducible over several repeats. (A) Mutant frequencies for lac G·C→T·A transversions. The strains used were: NR13153 (dnaX+), NR13159 (dnaX+ dinB), NR16889 (dnaX+ ΔpolB), NR17225 (dnaX+ ΔpolB dinB), NR16878 (dnaX+ polBex), NR17223 (dnaX+ polBex dinB), NR13256 (dnaX36); NR13274 (dnaX36, dinB); NR16159 (dnaX36, ΔpolB); NR16163 (dnaX36 ΔpolB dinB), NR16108 (dnaX36 polBex), and NR16116 (dnaX36 polBex dinB). The frequencies ± the SE values were 17.2 ± 1.3, 6.3 ± 0.8, 16.8 ± 1.9, 7.9 ± 0.5, 206 ± 13, 119 ± 8, 135 ± 10, 35 ± 5, 643 ± 86, 22 ± 2, 2,210 ± 239, and 1,460 ± 168, respectively. (B) Mutant frequencies for lac A·T→T·A transversions. The strains used were NR16226 (dnaX+), NR16228 (dnaX+ dinB), NR16890 (dnaX+ ΔpolB), NR17226 (dnaX+ ΔpolB dinB), NR16879 (dnaX+ polBex), NR17224 (dnaX+ polBex dinB), NR16246 (dnaX36), NR16248 (dnaX36 dinB), NR16176 (dnaX36 ΔpolB), NR16183 (dnaX36 ΔpolB dinB), NR16169 (dnaX36 polBex), and NR16173 (dnaX36 polBex, dinB). The mutant frequencies ± the SE were 1.1 ± 0.1, 1.1 ± 0.2, 1.6 ± 0.2, 1.7 ± 0.2, 8.3 ± 0.8, 3.8 ± 0.7, 11.9 ± 1.4, 6.9 ± 1.1, 105 ± 14, 6.9 ± 1.2, 263 ± 27, and 189 ± 20, respectively. (C) Mutant frequencies for trpE9777 reversion. Strains used were as described in panel A. The frequencies ± the SE were 313 ± 19, 452 ± 57, 515 ± 28, 289 ± 24, 404 ± 37, 309 ± 19, 3,770 ± 350, 403 ± 49, 9,210 ± 2,268, 633 ± 98, 4,790 ± 487, and 4,280 ± 332, respectively. (D) Mutant frequencies for Rifr. The strains used were as described in panel A. The frequencies ± the SE were 343 ± 35, 374 ± 13, 484 ± 24, 327 ± 19, 500 ± 63, 345 ± 14, 707 ± 48, 617 ± 44, 714 ± 92, 500 ± 71, 3,800 ± 550, and 2,800 ± 218, respectively.
FIG. 3.
FIG. 3.
Diagram depicting the various ways by which a Pol III misinsertion error may be processed in an error-free or error-prone manner. The scheme includes the competition between polymerases that may ensue upon dissociation of a Pol III HE from the mismatch (line 3 or 4), as well as the proposed τ-dependent mode of error removal (61) (line 6). Note that Pol III, while initially unable to proofread the error in the stalled state, may do so effectively upon rebinding, since the latter may occur preferentially using the exonuclease active site (30, 59, 60).
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