Mutator and Antimutator Effects of the Bacteriophage P1hotGene Product

Abstract

The Hot (homolog of theta) protein of bacteriophage P1 can substitute for the Escherichia coli DNA polymerase III subunit, as evidenced by its stabilizing effect on certain dnaQ mutants that carry an unstable polymerase III proofreading subunit (antimutator effect). Here, we show that Hot can also cause an increase in the mutability of various E. coli strains (mutator effect). The hot mutator effect differs from the one caused by the lack of . Experiments using chimeric /Hot proteins containing various domains of Hot and along with a series of point mutants show that both N-and C-terminal parts of each protein are important for stabilizing the subunit. In contrast, the N-terminal part of Hot appears uniquely responsible for its mutator activity.The Escherichia coli chromosome is replicated with high efficiency and fidelity by DNA polymerase (Pol) III holoenzyme (HE), a large dimeric multisubunit enzyme complex that simultaneously copies the leading and lagging strands at the replication fork (for reviews, see references 15 and 24). HE contains two core polymerase subassemblies, each composed of three separate subunits, ␣, ε, and , which are tightly bound in the linear order ␣-ε-. The ␣ subunit (dnaE gene product) (135 kDa) is the polymerase, while the ε subunit (dnaQ gene product) (28.5 kDa) is the 3Ј35Ј exonucleolytic activity that functions as a proofreader for replication errors.The precise function of the subunit (holE gene product) (8 kDa) within the Pol III core is less clear. binds tightly to the ε subunit and does not seem to interact with the ␣ subunit (2, 32). Strains lacking (⌬holE strains) are viable, indicating that the subunit is not essential (31). On the other hand, our laboratory has shown that such deletion strains possess a mutator phenotype in mismatch repair-defective strains, suggesting that may have a positive effect on the accuracy of DNA replication, likely through its effect on the ε proofreading activity (33). This fidelity role is consistent with the increase in the ε exonuclease activity observed in an in vitro exonuclease assay in the presence of (32). In addition, large effects of the deletion of were observed in strains containing an impaired or unstable ε subunit (33). For example, the mutability of the temperature-sensitive dnaQ49 mutator strain was increased nearly 1,000-fold upon the loss of (⌬holE strain). Based on these and other results, it was postulated (33) that the subunit fulfills a general stabilizing role for the intrinsically unstable ε subunit (7, 10, 12).appears to be well preserved throughout the enterobacteria, suggestive of a meaningful role for the protein. Homologs of have also been found, surprisingly, to be encoded by two conjugative plasmids as well as by bacteriophage P1 (3). In experiments with this P1 homolog produced by the P1 hot gene (homolog of theta) (22), we showed that the resulting Hot protein can substitute for in certain dnaQ mutators such as dnaQ49, as it was capable of reducing the high mutability of a dnaQ49 ⌬holE strain (3). In fact, for d...

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

“…Interestingly, at the same time it is capable of strongly increasing the mutability of another dnaQ mutant, dnaQ930 [13]. The precise reason for this mutator effect of Hot is unknown, but likely reflects a slightly different interaction of θ and Hot with ε that exacerbates the dnaQ930 defect (H98Y) [13,20]. This dual phenomenology permits a convenient test for expression of hot from the P1 prophage by checking whether these mutator/antimutator effects can be reproduced by a resident prophage.…”

“…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].…”

“…coli strains NR17116 (dnaQ49 zae-502::Tn10), NR16328 (dnaQ930 zae-502::Tn10), NR16320 (ΔholE dnaQ923 zae-502::Tn10), and NR16329 (ΔholE dnaQ930 zae-502::Tn10) are derivatives of strain MG1655 [16] and have been described [13]. KA796 (ara, thi, Δprolac) has also been described [17].…”

“…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]. The precise reason for this mutator effect of Hot is unknown, but likely reflects a slightly different interaction of θ and Hot with ε that exacerbates the dnaQ930 defect (H98Y) [13,20].…”

“…This dual phenomenology permits a convenient test for expression of hot from the P1 prophage by checking whether these mutator/antimutator effects can be reproduced by a resident prophage. We created lysogens of P1 and P1 Δhot of strains NR16320 (ΔholE dnaQ923) and NR16329 (ΔholE dnaQ930) [13], selecting for chloramphenicol-resistant derivatives at 30°, and measured the frequency of rifampicin-resistant mutants. In Fig.…”