Competition of Escherichia coli DNA polymerases I, II and III with DNA Pol IV in stressed cells

Abstract

Escherichia coli has five DNA polymerases, one of which, the low-fidelity Pol IV or DinB, is required for stress-induced mutagenesis in the well-studied Lac frameshift-reversion assay. Although normally present at approximately 200 molecules per cell, Pol IV is recruited to acts of DNA double-strand-break repair, and causes mutagenesis, only when at least two cellular stress responses are activated: the SOS DNA-damage response, which upregulates DinB approximately 10-fold, and the RpoS-controlled general-stress response, which upregulates Pol IV about 2-fold. DNA Pol III was also implicated but its role in mutagenesis was unclear. We sought in vivo evidence on the presence and interactions of multiple DNA polymerases during stress-induced mutagenesis. Using multiply mutant strains, we provide evidence of competition of DNA Pols I, II and III with Pol IV, implying that they are all present at sites of stress-induced mutagenesis. Previous data indicate that Pol V is also present. We show that the interactions of Pols I, II and III with Pol IV result neither from, first, induction of the SOS response when particular DNA polymerases are removed, nor second, from proofreading of DNA Pol IV errors by the editing functions of Pol I or Pol III. Third, we provide evidence that Pol III itself does not assist with but rather inhibits Pol IV-dependent mutagenesis. The data support the remaining hypothesis that during the acts of DNA double-strand-break (DSB) repair, shown previously to underlie stress-induced mutagenesis in the Lac system, there is competition of DNA polymerases I, II and III with DNA Pol IV for action at the primer terminus. Up-regulation of Pol IV, and possibly other stress-response-controlled factor(s), tilt the competition in favor of error-prone Pol IV at the expense of more accurate polymerases, thus producing stress-induced mutations. This mutagenesis assay reveals the DNA polymerases operating in DSB repair during stress and also provides a sensitive indicator for DNA polymerase competition and choice in vivo.

Figure 1. Representative examples of stress-induced mutagenesis data.

See Table 1 for quantification of mutation rates from multiple experiments. (A) Loss of Pol II increases mutagenesis both in Lex⁺ and lexA(Def) (SOS-constitutive) cells. (B) The hypermutagenesis observed in the ΔpolB strain is completely dinB-dependent. (C) Deficiency of Pol I increases Pol IV-dependent mutagenesis both in Lex⁺ and lexA(Ind⁻) (SOS-uninducible) cells. (D) Loss of the polymerase domain of Pol I in the polA6 mutant increases mutagenesis. (E) Loss of Pol I and Pol II increases Pol IV-dependent mutagenesis more than the absence of either Pol I or Pol II alone. (F) Data from (E), but with the y-axis expanded. (G) The dnaE915 gene product decreases mutagenesis both in Lex⁺ and lexA(Def) (SOS-constitutive) cells. (H) Proofreading-defective Pol III (ΔdnaQ) does not increase stress-induced mutagenesis. Therefore, DNA Pol III neither makes stress-induced mutations nor proofreads Pol IV errors. (I) Deletion of umuDC does not change stress-induced frameshift reversion in wild-type or dinB10 cells.

Figure 2. Pol IV protein levels are not increased SOS-independently in cells carrying the ΔpolB, polATS or dnaE915 mutations.

Numbers represent densitometer readings of band intensity normalized to dinB⁺. Strains, from left to right: SMR4562, SMR5889, SMR10308, SMR868, PH306, SMR5400, SMR8913, SMR7767. Two separate experiments gave similar results.

Figure 3. Model for the mechanism of stress-induced frameshift reversion.

(Modified from , .) Double-strand ends (DSEs), formed by replication-fork collapse upon encountering a single-strand nick, are processed by RecBCD to form single-strand DNA. RecA promotes recombination with homologous DNA to initiate repair. About 40% of stationary-phase cells have two chromosomes , making a sister DNA molecule a probable repair partner. The 3′-invading end in the D-loop recombination intermediate primes DNA synthesis (dashed lines), and the structure is resolved by RuvABC to yield a repaired molecule. DNA synthesis can be either high- or low-fidelity, depending on the DNA polymerase(s) used: High-fidelity synthesis results from Pols I, II or III, whereas low-fidelity synthesis [yielding localized frameshift mutations (X)] results from Pol IV. Upregulation of dinB by SOS and RpoS stress responses results in more Pol IV molecules per cell, and possibly a more competitive Pol IV, which successfully competes with Pols I, II, and III for the sites of DNA synthesis during DSB repair, allowing Pol IV-dependent frameshift mutagenesis.