A mutant of Escherichia coli K12 deficient in the 5′–3′ exonucleolytic activity of DNA polymerase I

Heijneker, H. L.; Ellens, D. J.; Tjeerde, R.; Glickman, Barry W.; Dorp, Bob Van; Pouwels, Peter H.

doi:10.1007/bf00267167

Cited by 55 publications

(13 citation statements)

References 27 publications

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“…Most relevant, is the polA107 allele [40] that encodes a Y77C mutant of pol I (Fig. 2A) [41] lacking 5′→3′ exo/endonuclease activity [42], which is viable in the recA730 background [39]. To assay the role that the 5′→3′ exo/endonuclease of pol I has in ribonucleotide repair, we transduced the polA107 allele that had previously been linked to zih219 ::Tn 10 into a recA730 lexA51 (Def) Δ dinB Δ umuDC Δ mutL strain background (Table 1).…”

Section: Resultsmentioning

confidence: 99%

Investigating the mechanisms of ribonucleotide excision repair in Escherichia coli

Vaisman

McDonald

Noll

et al. 2014

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

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Low fidelity Escherichia coli DNA polymerase V (pol V/UmuD′2C) is best characterized for its ability to perform translesion synthesis (TLS). However, in recA730 lexA(Def) strains, the enzyme is expressed under optimal conditions allowing it to compete with the cell’s replicase for access to undamaged chromosomal DNA and leads to a substantial increase in spontaneous mutagenesis. We have recently shown that a Y11A substitution in the “steric gate” residue of UmuC reduces both base and sugar selectivity of pol V, but instead of generating an increased number of spontaneous mutations, strains expressing umuC_Y11A are poorly mutable in vivo. This phenotype is attributed to efficient RNase HII-initiated repair of the misincorporated ribonucleotides that concomitantly removes adjacent misincorporated deoxyribonucleotides. We have utilized the ability of the pol V steric gate mutant to promote incorporation of large numbers of errant ribonucleotides into the E. coli genome to investigate the fundamental mechanisms underlying ribonucleotide excision repair (RER). Here, we demonstrate that RER is normally facilitated by DNA polymerase I (pol I) via classical “nick translation”. In vitro, pol I displaces 1–3 nucleotides of the RNA/DNA hybrid and through its 5′→3′ (exo/endo) nuclease activity releases ribo- and deoxyribonucleotides from DNA. In vivo, umuC_Y11A-dependent mutagenesis changes significantly in polymerase-deficient, or proofreading-deficient polA strains, indicating a pivotal role for pol I in ribonucleotide excision repair (RER). However, there is also considerable redundancy in the RER pathway in E. coli. Pol I’s strand displacement and FLAP- exo/endonuclease activities can be facilitated by alternate enzymes, while the DNA polymerization step can be assumed by high-fidelity pol III. We conclude that RNase HII and pol I normally act to minimize the genomic instability that is generated through errant ribonucleotide incorporation, but that the “nick-translation” activities encoded by the single pol I polypeptide can be undertaken by a variety of back-up enzymes.

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Section: Resultsmentioning

confidence: 99%

Investigating the mechanisms of ribonucleotide excision repair in Escherichia coli

Vaisman

McDonald

Noll

et al. 2014

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

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“…This difference may reflect the enzymic discreteness of these two activities, which can be separated by proteolytic cleavage of polymerase I in vitro (15,16). Glickman et al (17) and Heijneker et al (18) have very recently observed a mutation (polA '107) th4t may be similar to pQlAexl; however, the extent of the enzymatic defect is not completely clear, and the polA '107 mutation is not conditionally lethal.…”

Section: Resultsmentioning

confidence: 99%

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

Konrad

Lehman

1974

Proc. Natl. Acad. Sci. U.S.A.

140

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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 ...

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“…However, our data should not necessarily be taken to imply that this part of the 5 H -3 H exonuclease domain plays no role in the reaction. Other residues in this region (Arg70, Tyr77, Lys78, and Arg81 on the Pol I sequence) are invariant in the alignment of 18 sequences and the Y77C mutation (polA107 allele) inactivates the 5 H -3 H exonuclease (Heijneker et al, 1973;Joyce et al, 1985). In Taq DNA polymerase this region is disordered with 21 amino acid residues not visible in the structure (Kim et al, 1995); in T4 RNase H nine amino acid residues are missing (Mueser et al, 1996).…”

Section: Mutational Analysis Of the Conserved Carboxylatesmentioning

confidence: 95%

Biochemical and mutational studies of the 5′-3′ exonuclease of DNA polymerase I of Escherichia coli

Derbyshire

et al. 1997

Journal of Molecular Biology

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A mutant of Escherichia coli K12 deficient in the 5′–3′ exonucleolytic activity of DNA polymerase I

Cited by 55 publications

References 27 publications

Investigating the mechanisms of ribonucleotide excision repair in Escherichia coli

Investigating the mechanisms of ribonucleotide excision repair in Escherichia coli

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

Biochemical and mutational studies of the 5′-3′ exonuclease of DNA polymerase I of Escherichia coli

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