A Conditional Lethal Mutant of Escherichia coli K12 Defective in the 5′ → 3′ Exonuclease Associated with DNA Polymerase I

Abstract

A mutant strain of E. coli, initially identified by an abnormally high frequency of recombination, has been found to be defective in the 5' -. 3' exonuclease associated with DNA polymerase I, but not in the polymerase activity. This defect is tolerated at 300, but is lethal at 43°. Like other polymerase I mutants, the strain is unusually sensitive to methyl methanesulfonate and to ultraviolet irradiation; it is also unable to support the growth of phage X defective in general recombination, and shows a reduced rate of joining of 10S "Okazaki fragments." These results demonstrate that a functional DNA polymerase I is essential for normal growth and viability in E. coli K12.Escherichia coli K12 strains defective in the structural gene for DNA polymerase I (polA) have been extensively described (1-5). These mutants are abnormally sensitive to ultraviolet irradiation and to the alkylating agent methyl methanesulfonate, and show retarded joining of the 10S "Okazaki fragments" (6)(7)(8). A recent analysis of partially purified fractions derived from..several of the polA mutants has demonstrated that although they contain much reduced levels of DNA polymerase I activity the 5' --3' exonuclease associated with the polymerase I is present in nearly normal amounts (9).We have found that polA mutants are also characterized by an increased frequency of recombination at least under some conditions (7). By screening mutagenized cells for this phenotype, we have isolated a novel polA mutant (polAexl) that we describe in this paper. This mutant has a normal level of polymerase I activity but a greatly reduced level of 5' -_ 3' exonuclease. Unlike other polA mutants, it is conditionally lethal. MATERIALS AND METHODSBacterial and Phage Strains. All bacterial strains are derived from E. coli K12. Their origin and genotypes are given in Table 1. Phage X defective in general recombination (A red 3) was provided by D. Freifelder.Microbiological Procedures. Pivir phage stocks were prepared and used for transduction, and Hfr crosses were performed as described by Miller (10). Sensitivity to ultraviolet irradiation was determined as follows. Cells grown at 30°in H broth (11) to A595 = 0.5 were chilled, harvested, and resuspended in an equal volume of chilled M63 medium (10); 10 ml of a 5-fold dilution of this suspension in a glass petri plate was irradiated (25 ergs per mm2 per sec) with a General Electric germicidal lamp; and samples were removed at various times, diluted in chilled H broth, and plated on yeast extract-tryptone plates (10) that were incubated at 300 in the dark. Sensitivity to methyl methanesulfonate was determined on fresh yeast extract-tryptone plates supplemented with 0.04% methyl methanesulfonate. Cultures were treated with ethyl methanesulfonate as described by Miller (10). Lactosetetrazolium indicator plates were prepared according to Miller (10). Ability to form plaques of phage A was determined on tryptone plates as described by Miller (10). Unless otherwise noted, E. coli polAexl strains were grown at ...

DnaA protein stimulates polA gene expression in Escherichia coli

“…It is encoded by the polA gene located at 87.5 min on the E. coli genetic map (Kelley and Grindley, 1976). Several mutations in the polA gene have been isolated and characterized (De Lucia and Cairns, 1969;Monk and Kinross, 1972;Konrad and Lehman, 1974;Joyce and Grindley, 1984;Joyce etal., 1985). They revealed Received 4 October, 1996;revised 18 December, 1996; accepted 3 January, 1997.…”

Symbiotic Plasmid Rearrangement in a Hyper-recombinant Mutant of Rhizobium leguminosarum biovar phaseoli

“…The CFN2300 phenotype presented here is similar to that of certain E.coli mutants affected in DNA replication or repair (De Lucia & Cairns, 1969;Gross & Gross, 1969;Kinsbury & Helinski, 1973;Lehman & Chien, 1973;Konrad & Lehman, 1974;Chase & Richardson, 1977;Drlica, 1984;Bassett & Kushner, 1985;Charon et al, 1985;Smith, 1988), but the precise defect of strain CFN2300 remains to be established. Syrn plasmid deletion in the genetic background of strain CFN2300 Strain CFN2300 has a higher frequency of homologous genetic recombination than strain CFN2315 (Table 4).…”

“…Mutations in the genes coding for DNA polymerase I (poZA), exonuclease VII (xsc), helicase I1 (uvrD), DNA ligase (Zig), DNA adenine methylase (dam) and deoxyuridine triphosphatase (dut) genes have been selected on the basis of higher frequency of genetic recombination (Konrad & Lehman, 1974;Konrad, 1977;Chase & Richardson, 1977;Arthur & Lloyd, 1980). We describe here the isolation of an R. leguminosarum biovarphaseoli CFN23 derivative cured of an indigenous plasmid (p23a) different from the Sym plasmid.…”

Saccharomyces cerevisiae RAD27 complements its Escherichia coli homolog in damage repair but not mutation avoidance

“…The rad27 null mutant strains arrest in G2/M at 37 ∞ C, grow slowly at 30 ∞ C, and exhibit increased plasmid loss (Reagan et al 1995). The mutant deficient in E. coli polA 5' 3' exonuclease also exhibits temperature-sensitive lethality (Konrad and Lehman 1974). These phenotypes suggest the importance of flapendonuclease for DNA replication.…”

“…The nuclease activity of Rad27/Fen-1 removes RNA primers made during lagging-strand DNA synthesis (Bambara et al 1997). In Escherichia coli and other bacteria, the removal of RNA primers is performed by 5' 3' exonuclease activity of DNA polymerase I (PolI), encoded by the polA gene (Konrad and Lehman 1974;Kornberg and Baker 1992), which is a homolog of Rad27/Fen-1 nucleases (Robins et al 1994). …”