Mutator mutants of Escherichia coli carrying a defect in the DNA polymerase III τ subunit

Abstract

SummaryTo investigate the possible role of accessory subunits of Escherichia coli DNA polymerase III holoenzyme (HE) in determining chromosomal replication fidelity, we have investigated the role of the dnaX gene. This gene encodes both the t and g subunits of HE, which play a central role in the organization and functioning of HE at the replication fork. We find that a classical, temperature-sensitive dnaX allele, dnaX36 , displays a pronounced mutator effect, characterized by an unusual specificity: preferential enhancement of transversions and -1 frameshifts. The latter occur predominantly at non-run sequences. The dnaX36 defect does not affect the g subunit, but produces a t subunit carrying a missense substitution (E601K) in its C-terminal domain (domain V) that is involved in interaction with the Pol III a subunit. A search for new mutators in the dnaX region of the chromosome yielded six additional dnaX mutators, all carrying a specific t subunit defect. The new mutators displayed phenotypes similar to dnaX36 : strong enhancement of transversions and frameshifts and only weak enhancement for transitions. The combined findings suggest that the t subunit of HE plays an important role in determining the fidelity of the chromosomal replication, specifically in the avoidance of transversions and frameshift mutations.

Dpb2p, a Noncatalytic Subunit of DNA Polymerase ε, Contributes to the Fidelity of DNA Replication in Saccharomyces cerevisiae

“…Our results indicate that noncatalytic replicase subunits may play an important role in maintaining the fidelity of DNA replication in eukaryotes, as has been shown previously in prokaryotic systems (Pham et al 2006).…”

“…One important factor is the status of the replication checkpoint, since pol3-01 and pol2-4 have similar mutation rates in checkpoint-defective dun1D mutants (Datta et al 2000). There are other examples suggesting that noncatalytic DNA polymerase subunits may contribute to polymerase fidelity (Araki et al 1991b;Huang et al 2002;Pham et al 2006). The absence of Pol32p, a nonessential subunit of Pol d HE of S. cerevisiae, causes increases in genomic deletions of sequences flanked by short direct repeats (Huang et al 2002).…”

“…In addition, deletion of the Dpb3p subunit of Pol e HE (dpb3D) elevated the spontaneous-reversion rate by factors 2.2, 2.6, and 20 at his1-7, ade2-1, and lys1-1, respectively (Araki et al 1991b). In Escherichia coli, mutations in the dnaX gene, encoding the t-subunit of DNA Pol III HE, lead to a mutator phenotype (Pham et al 2006).…”

Role of Accessory DNA Polymerases in DNA Replication in Escherichia coli : Analysis of the dnaX36 Mutator Mutant

“…These studies revealed that the temperature-sensitive allele dnaX36, encoding a mutant subunit containing a Glu-to-Lys change at residue 601 in domain V (4), displayed elevated mutation rates (61). This mutator phenotype suggested that one additional function of is to promote the fidelity of the chromosomal replication complex.…”

“…Previously, our laboratory has investigated the properties of certain E. coli dnaX mutants (61). These studies revealed that the temperature-sensitive allele dnaX36, encoding a mutant subunit containing a Glu-to-Lys change at residue 601 in domain V (4), displayed elevated mutation rates (61).…”