DNA replication fidelity in Escherichia coli: a multi-DNA polymerase affair

Abstract

High accuracy (fidelity) of DNA replication is important for cells to preserve the genetic identity and to prevent the accumulation of deleterious mutations. The error rate during DNA replication is as low as 10(-9) to 10(-11) errors per base pair. How this low level is achieved is an issue of major interest. This review is concerned with the mechanisms underlying the fidelity of the chromosomal replication in the model system Escherichia coli by DNA polymerase III holoenzyme, with further emphasis on participation of the other, accessory DNA polymerases, of which E. coli contains four (Pols I, II, IV, and V). Detailed genetic analysis of mutation rates revealed that (1) Pol II has an important role as a back-up proofreader for Pol III, (2) Pols IV and V do not normally contribute significantly to replication fidelity, but can readily do so under conditions of elevated expression, (3) participation of Pols IV and V, in contrast to that of Pol II, is specific to the lagging strand, and (4) Pol I also makes a lagging-strand-specific fidelity contribution, limited, however, to the faithful filling of the Okazaki fragment gaps. The fidelity role of the Pol III τ subunit is also reviewed.

Fig. 1

General scheme indicating how three serial fidelity steps during chromosomal replication can produce the low error rate of ~10⁻¹⁰ errors per base per round of replication. The steps are: (a) discrimination by the polymerase against inserting an incorrect base, (error rate ~10⁻⁵); (b) proofreading (editing) of misinserted bases (T·T, example) by the 3'→5' exonuclease associated with the polymerase (escape rate ~10⁻²); and (c) removal of remaining mismatches (G·G, example) by postreplicative DNA Mismatch Repair (MMR) (escape rate ~10⁻³).

Fig. 2

A model of the E. coli DNA polymerase III holoenzyme (HE) at the chromosomal replication fork, synthesizing, simultaneously, leading and lagging strands in, respectively, continous (leading strand) and discontinuous (lagging strand) fashion. The αεθ complex represents the Pol III core, in which α is the polymerase, ε is the exonuclease (proofreading) subunit, and θ is a stabilizing subunit. See text for details. Not shown is the DnaG primase, which, in association with the DnaB helicase, produces lagging-strand RNA primers supporting the discontinous synthesis in this strand. The γ complex (γδδ'χψ) conducts the cycling (loading and unloading) of the β₂ processivity clamps, which is particularly important for the cycling of the polymerase in the lagging strand. Recent studies have suggested that the relevant form of the DnaX assembly (τ₂γδδ'χψ as shown here) may be τ₃δδ'χψ (noting that γ and τ are both products of the dnaX gene and differ only in their C-termini). As τ contains the extra C-terminal extension that mediates the τ-α interaction, the τ₃δδ'χψ-containing HE is capable of binding a third Pol III core (McInerney et al., 2007; Reyes-Lamothe et al., 2010; Georgescu et al., 2012). This third Pol III core (not shown) may participate in polymerase switching and hence contribute to the chromosomal replication process (see text).

Fig. 3

The five DNA polymerases of E. coli and some of their relevant properties.

Fig. 4

Proposed scheme for the involvement of E. coli DNA polymerases in error production and prevention at the replication fork, as based on the work described in this review. Events start when Pol III commits a misinsertion error (indicated as T:T or A:A) in either DNA strand. While Pol III may handle the mismatch by itself by either extending the mismatch, yielding a potential mutation, or by removing it using its proofreading activity (not shown), in a fraction of cases Pol III may dissociate from the mismatch, leading to a possible polymerase exchange (arrows). The data suggest that, in both strands, Pol II is the primary enzyme for such a polymerase exchange, leading to removal of the terminal mismatch by the 3' exonuclease of Pol II (back-up proofreading function). When dissociation occurs in the lagging strand, Pol IV and Pol V may also compete for the primer terminus, leading to extension of the mismatch and fixing the error as a potential mutation. In contrast, Pol I does not compete for the primer terminus in either strand. Instead, its fidelity function is limited to the error-free filling of the Okazaki fragment gaps. We have also indicated in the scheme the third Pol III core (shaded green) that may be present in HE if the indicated DnaX₃ assembly were composed of three τ subunits (McInerney et al., 2007; Reyes-Lamothe et al., 2010; Georgescu et al., 2012). If present, this third core might be expected to behave similarly to Pol II, leading, upon binding to the terminus, to mismatch removal (preferential binding with exonuclease site). See text for further details and justification.