dnaX36 Mutator of Escherichia coli : Effects of the τ Subunit of the DNA Polymerase III Holoenzyme on Chromosomal DNA Replication Fidelity

Abstract

The Escherichia coli dnaX36 mutant displays a mutator effect, reflecting a fidelity function of the dnaXencoded subunit of the DNA polymerase III (Pol III) holoenzyme. We have shown that this fidelity function (i) applies to both leading-and lagging-strand synthesis, (ii) is independent of Pol IV, and (iii) is limited by Pol II.The mechanisms by which organisms achieve a high accuracy of DNA replication are of ongoing interest. Replication of the chromosome of the bacterium Escherichia coli is performed by the DNA polymerase III (Pol III) holoenzyme (HE). HE is composed of 17 subunits (10 distinct), with an overall composition (␣ε) 2 ␤ 4 2 ␥␦␦Ј (27). It contains two (␣ε) polymerase core assemblies, one for the leading strand and one for the lagging strand. The ␣ subunit is the DNA polymerase, ε is the 3Ј 3 5Ј proofreading exonuclease, and is an ε-stabilizing factor (2, 17, 34). Within HE, a central role is played by the subunit ( 2 ), which has several important functions, including connecting the two polymerases, enabling coupled leadingand lagging-strand synthesis. For the fidelity of replication, most of the focus has been on the DNA polymerase III core, notably the polymerase and the proofreading subunit (26,28,29). However, it is clear that overall chromosomal fidelity is not simply a function of polymerase fidelity but also involves activities of other HE subunits-as evidenced by mutator effects associated with defects in such subunits (24, 30, 32)-and the participation of accessory DNA polymerases (1, 8, 9, 12-14, 23, 35), of which E. coli has four (Pol I, II, IV, and V).The present study is concerned with the fidelity role of the central subunit of HE, encoded by the dnaX gene, which also encodes the ␥ subunit (6). is the full-length product of the gene (643 amino acids), while the ␥ subunit is an early termination product (residues 1 to 430). Since and ␥ share the N-terminal protein sequence (domains I, II, and III), they share certain functions, such as the loading and unloading of the ␤-processivity clamps (27). However, the two additional domains (IV and V) that are present in permit it to perform certain unique functions. Specifically, domain IV contains the site of interaction with the DnaB helicase (10), which positively regulates the speed of the replication fork (4). Domain V contains the -␣ interaction site that enables HE to be dimeric (11). Domain V also controls the cycling of the lagging-strand polymerase, as it mediates the release of the Pol III core from its ␤ processivity clamp upon completion of Okazaki fragments (20, 21). Thus, is an important control element within HE that can influence polymerase behavior, and this may extend to HE fidelity.A fidelity role for the subunit was proposed based on observations of a distinct mutator activity for certain dnaX mutants (30). In particular, dnaX36 was informative since its defect (E601K) resides in domain V and hence only affects . Presumably, in the dnaX36 mutant, the -␣ interaction is altered, leading to the mutator effect. An additional ...

Defect of Dpb2p, a noncatalytic subunit of DNA polymerase ɛ, promotes error prone replication of undamaged chromosomal DNA in Saccharomyces cerevisiae

“…DNA polymerase III holoenzyme (Pol III HE), the major replicative DNA polymerase in Escherichia coli, is a large complex of 17 subunits [2,3]. It has been shown that some mutations in the dnaX gene which encodes the subunit of Pol III HE lead to a mutator phenotype [4][5][6]. Also in Saccharomyces cerevisiae the accessory subunits of the main replicases have been demonstrated to affect the fidelity of DNA replication.…”

Proofreading deficiency of Pol I increases the levels of spontaneous rpoB mutations in E. coli

“…In certain cases this contribution may lead to improved fidelity, when the accessory DNA polymerase is accurate (e.g., proofreading-proficient polymerases), while in other cases this may lead to lower fidelity when the polymerase is error-prone. Examples of both effects have been demonstrated [1–7]. …”

“…E. coli contains 5 different DNA polymerases of which DNA polymerase III is the main replicative polymerase responsible for chromosomal duplication (DNA polymerase III holoenzyme, HE). DNA polymerase II can play an important fidelity role by acting as a back-up proofreader for errors created by Pol III [1,7,10]. In contrast, Pol IV and Pol V, when present in amplified amounts, can reduce replication fidelity.…”

“…In each of these cases, it has been proposed that these DNA accessory polymerases can compete with Pol III HE for the growing point. This may occur particularly when progress of the replication fork is temporarily stalled by a Pol III-generated error (terminal mismatch) [1–3,7,10]. The precise mechanism of polymerase switching at the replication point is an active area of research.…”

Effect of dNTP pool alterations on fidelity of leading and lagging strand DNA replication in E. coli

“…It is attractive to speculate that improved lagging-strand fidelity results from the more dissociative properties of the polymerase in this strand, permitting more efficient proofreading (note that proofreading is essentially a dissociative process in which the primer terminus is moved from the polymerase active site to the distant exonuclease site). Alternatively, the dissociative character of the lagging-strand polymerase may lead to increased removal of the terminal mismatch by the exonuclease of Pol II, which has been shown to proofread for Pol III under some conditions [12, 13, 34, 35] or by a third copy of Pol III that has been proposed to be present at the replication fork [36]. …”