The Bacteriophage P1hotGene Product Can Substitute for theEscherichia coliDNA Polymerase III θ Subunit

Abstract

The subunit (holE gene product) of Escherichia coli DNA polymerase (Pol) III holoenzyme is a tightly bound component of the polymerase core. Within the core (␣--), the ␣ and subunits carry the DNA polymerase and 3 proofreading functions, respectively, while the precise function of is unclear. holE homologs are present in genomes of other enterobacteriae, suggestive of a conserved function. Putative homologs have also been found in the genomes of bacteriophage P1 and of certain conjugative plasmids. The presence of these homologs is of interest, because these genomes are fully dependent on the host replication machinery and contribute few, if any, replication factors themselves. To study the role of these homologs, we have constructed an E. coli strain in which holE is replaced by the P1 homolog, hot. We show that hot is capable of substituting for holE when it is assayed for its antimutagenic action on the proofreading-impaired dnaQ49 mutator, which carries a temperature-sensitive subunit. The ability of hot to substitute for holE was also observed with other, although not all, dnaQ mutator alleles tested. The data suggest that the P1 hot gene product can substitute for the subunit and is likely incorporated in the Pol III complex. We also show that overexpression of either or Hot further suppresses the dnaQ49 mutator phenotype. This suggests that the complexing of dnaQ49-with is rate limiting for its ability to proofread DNA replication errors. The possible role of hot for bacteriophage P1 is discussed.Escherichia coli DNA polymerase III holoenzyme (HE) is the enzyme responsible for the faithful duplication of the bacterial chromosome. HE is a multisubunit, dimeric complex that replicates simultaneously the leading and lagging strands at the replication fork (for a review, see reference 31). Each half of the dimeric complex contains a polymerase (Pol) III core unit consisting of three subunits, ␣, ε, and , which are tightly bound in the linear order ␣-ε-. Of these, ␣ (135 kDa) is the DNA polymerase (dnaE gene product), ε (28 kDa) is the exonucleotidic proofreader responsible for editing polymerase insertion errors (dnaQ gene product), and (8 kDa) is a small subunit whose function is not well defined (holE gene product). The HE (17 subunits total, 10 distinct) further consists of the two ␤-clamps (one for each core) that serve to tether the polymerase to the DNA and the DnaX complex ( 2 ␥␦␦Ј), which is responsible for connecting the two polymerases and for loading and unloading the ␤-clamp in the discontinuously synthesized lagging strand. The precise mechanisms by which HE is capable of replicating the bacterial chromosome with high speed and high accuracy are the subject of active investigation (21, 31, 33, 52).Our laboratory has been interested in the roles and mechanisms of the ε and subunits within the Pol III core, particularly with respect to their contributions to the fidelity of the replication process (37,(48)(49)(50). ε is a critical subunit with at least dual functions. It is a fidelity subunit whose...

The bacteriophage P1 hot gene, encoding a homolog of the E. coli DNA polymerase III θ subunit, is expressed during both lysogenic and lytic growth stages

“…By implication, an answer to this question is likely to bear on the precise role of θ in E. coli. Genetic studies have shown that hot, when cloned and expressed from the holE promoter on either a low-copy plasmid or the E. coli chromosome, is able to complement a ΔholE defect, reducing, for example, the ΔholE dnaQ49 mutator effect by some 1000-fold [9]. NMR structural studies on both θ and Hot revealed the two proteins to have near identical structures [10][11][12], further consistent with Hot being a fully functional homolog of θ. Alignment of the two proteins shows them to be approximately 48-53% identical (60-66% similar), which increases to 70 and 80%, respectively, when considering the structured core of the molecules [13].…”

“…Previously we also showed that hot, when expressed from a plasmid, was capable of competing with θ for incorporation into Pol III [9,13]. Therefore, we also tested if expression from the P1 prophage is sufficient to compete with θ.…”

“…KA796 (ara, thi, Δprolac) has also been described [17]. P1 bacteriophage c1-100 Tn9 used throughout this study has been described [6,9]. Strains BW25113 and BT430 [pCP20] and plasmids pKD4 and pKD46, all used for deletion mutagenesis of P1 by the method of Datsenko and Wanner [18], were obtained from the E. coli Genetic Stock Center (Yale University).…”

“…1B). To check for a possible requirement for hot in the absence of the host θ subunit, we also investigated the various properties of the Δhot phage in the ΔholE strain NR13104 [9]. No significant differences between the Δhot and hot + phages were found (data not shown).…”

“…We made use of our previous observations that Hot, when cloned and expressed from a low-copy plasmid, is capable of strongly reducing the mutability of several unstable dnaQ mutator mutants, such as dnaQ49 or dnaQ923 [9]. Interestingly, at the same time it is capable of strongly increasing the mutability of another dnaQ mutant, dnaQ930 [13].…”