Role of Escherichia coli DNA polymerase I in chromosomal DNA replication fidelity

We have investigated the possible role of Escherichia coli DNA polymerase (Pol) I in chromosomal replication fidelity. This was done by substituting the chromosomal polA gene by the polAexo variant containing an inactivated 3′→5′ exonuclease, which serves as a proofreader for this enzyme's misinsertion errors. Using this strain, activities of Pol I during DNA replication might be detectable as increases in the bacterial mutation rate. Using a series of defined lacZ reversion alleles in two orientations on the chromosome as markers for mutagenesis, 1.5‐ to 4‐fold increases in mutant frequencies were observed. In general, these increases were largest for lac orientations favouring events during lagging strand DNA replication. Further analysis of these effects in strains affected in other E. coli DNA replication functions indicated that this polAexo mutator effect is best explained by an effect that is additive compared with other error‐producing events at the replication fork. No evidence was found that Pol I participates in the polymerase switching between Pol II, III and IV at the fork. Instead, our data suggest that the additional errors produced by polAexo are created during the maturation of Okazaki fragments in the lagging strand.     

Novel blunt-end addition reactions catalyzed by DNA polymerase I of Escherichia coli

DNA polymerase I (Klenow fragment) of Escherichia coli catalyzes the addition of deoxynucleotides to 3' hydroxyl termini of blunt-ended DNA fragments. The product of the reaction, which we call +1 addition, is found only in very low yield under conditions that permit editing by the 3'----5' exonuclease activity of the wild-type polymerase. A mutant form of the Klenow fragment that lacks detectable 3'----5' exonuclease activity shows an elevated accumulation of the +1 addition product. The mutant enzyme can use any one of the four dNTPs to carry out the reaction when each precursor is provided individually. However, in the presence of all four dNTPs the addition of dATP is strongly preferred. Suppression of the editing function of the wild-type polymerase through the use of high concentrations of exogenous deoxynucleoside monophosphates also results in a significant increase in the amount of +1 addition product formed. The presence of a high dNMP concentration also alters the specificity of the nucleotide addition reaction carried out by the wild-type enzyme. Thus, in addition to dATP, the dNTP which is complementary to the exogenous deoxynucleoside monophosphate, is also used in the +1 addition reaction. A similar effect of dNMPs on the specificity of nucleotide addition was obtained with the mutant Klenow fragment. These results define two pathways for the +1 addition reaction: one that does not require coding information from the DNA template and a second in which coding information is provided by the exogenous dNMP.     

Methylation-dependent DNA synthesis in Escherichia coli mediated by DNA polymerase I.

An in vitro system was used to study DNA synthesis in lysates of Escherichia coli cells which had been grown in the presence of ethionine. Such lysates showed a reduced capacity to incorporate [3H]TTP into high-molecular-weight material. Activity could be restored by incubation with S-adenosyl methionine and ATP. S-adenosyl methionine-reactivated TTP incorporation required the presence of DNA polymerase I, ATP, and all four deoxyribonucleotide triphosphates. DNA polymerase III was not required.     

DNA polymerase II as a fidelity factor in chromosomal DNA synthesis in Escherichia coli

Escherichia coli DNA polymerase III holoenzyme (HE) is the main replicase responsible for replication of the bacterial chromosome. E. coli contains four additional polymerases, and it is a relevant question whether these might also contribute to chromosomal replication and its fidelity. Here, we have investigated the role of DNA polymerase II (Pol II) (polB gene product). Mismatch repair-defective strains containing the polBex1 allele--encoding a polymerase-proficient but exonucleolytically defective Pol II--displayed a mutator activity for four different chromosomal lac mutational markers. The mutator effect was dependent on the chromosomal orientation of the lacZ gene. The results indicate that Pol II plays a role in chromosomal replication and that its role is not equal in leading- versus lagging-strand replication. In particular, the role of Pol II appeared larger in the lagging strand. When combined with dnaQ or dnaE mutator alleles, polBex1 showed strong, near multiplicative effects. The results fit a model in which Pol II acts as proofreader for HE-produced misinsertion errors. A second role of Pol II is to protect mismatched 3' termini against the mutagenic action of polymerase IV (dinB product). Overall, Pol II may be considered a main player in the polymerase trafficking at the replication fork.     

Translesional synthesis past acetylaminofluorene-derived DNA adducts catalyzed by human DNA polymerase kappa and Escherichia coli DNA polymerase IV.

Human DNA polymerase kappa (pol kappa) has a sequence significantly homologous with that of Escherichia coli DNA polymerase IV (pol IV). We used a truncated form of human pol kappa (pol kappaDeltaC) and full-length pol IV to explore the miscoding properties of these enzymes. Oligodeoxynucleotides, modified site-specifically with N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) and N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-AF), were used as DNA templates in primer extension reactions that included all four dNTPs. Reactions catalyzed by pol kappaDeltaC were partially blocked one base prior to dG-AAF or dG-AF, and also opposite both lesions. At higher enzyme concentrations, a significant fraction of primer was extended. Analysis of the fully extended reaction product revealed incorporation of dTMP opposite dG-AAF, accompanied by much smaller amounts of dCMP, dAMP, and dGMP and some one- and two-base deletions. The product terminating 3' to the adduct site contained AMP misincorporated opposite dC. On templates containing dG-AF, dAMP, dTMP, and dCMP were incorporated opposite the lesion in approximately equal amounts, together with some one-base and two-base deletions. Steady-state kinetics analysis confirmed the results obtained from primer extension reactions catalyzed by pol kappa. In contract, primer extension reactions catalyzed by pol IV were blocked effectively by dG-AAF and dG-AF. At high concentrations of pol IV, full-length products were formed containing primarily one- or two-base deletions with dCMP, the correct base, incorporated opposite dG-AF. The miscoding properties of pol kappa observed in this study are consistent with mutational spectra observed when plasmid vectors containing dG-AAF or dG-AF are introduced into simian kidney cells [Shibutani, S., et al. (2001) Biochemistry 40, 3717-3722], supporting a model in which pol kappa plays a role in translesion synthesis past acetylaminofluorene-derived lesions in mammalian cells.     

Influence of divalent metal activator on the specificity of misincorporation during DNA synthesis catalyzed by DNA polymerase I of Escherichia coli

To test whether the identity of divalent metal activator affects the specificity of misincorporation during polymerization catalyzed by E. coli DNA polymerase I, we carried out the following procedure. A series of oligonucleotide primers, annealed at different positions along the lacZ region of bacteriophage M13mp9 DNA, were elongated in the presence of 3 of the 4 deoxynucleoside 5'-triphosphates (dNTPs) until one or a few misincorporations occurred in each elongated primer. The elongated primers (containing deoxynucleotide residues that had been misincorporated in the presence of either Mg2+ or Mn2+) were then isolated and sequenced by the 'dideoxy' chain termination method to determine the identity of deoxynucleoside monophosphates (dNMPs) that had been misincorporated at different template positions during the original 'minus' reactions, activated by Mg2+ or Mn2+. The results obtained by this approach revealed that both the type of misincorporation and the effect of substituting Mn2+ for Mg2+ depended on the nucleotide sequence of the template. At 40% of the template positions at which misincorporation was compared with both metal ions (8 out of 20), the identity of mispairs differed significantly for synthesis activated by Mn2+ versus Mg2+. Of these 8 sites, 4 exhibited increased transversions in the presence of Mn2+, while 4 exhibited decreased transversions with Mn2+.     

Highly tolerated amino acid substitutions increase the fidelity of Escherichia coli DNA polymerase I

Fidelity of DNA synthesis, catalyzed by DNA polymerases, is critical for the maintenance of the integrity of the genome. Mutant polymerases with elevated accuracy (antimutators) have been observed, but these mainly involve increased exonuclease proofreading or large decreases in polymerase activity. We have determined the tolerance of DNA polymerase for amino acid substitutions in the active site and in different segments of E. coli DNA polymerase I and have determined the effects of these substitutions on the fidelity of DNA synthesis. We established a DNA polymerase I mutant library, with random substitutions throughout the polymerase domain. This random library was first selected for activity. The essentiality of DNA polymerases and their sequence and structural conservation suggests that few amino acid substitutions would be tolerated. However, we report that two-thirds of single base substitutions were tolerated without loss of activity, and plasticity often occurs at evolutionarily conserved regions. We screened 408 members of the active library for alterations in fidelity of DNA synthesis in Escherichia coli expressing the mutant polymerases and carrying a second plasmid containing a beta-lactamase reporter. Mutation frequencies varied from 1/1000- to 1000-fold greater compared with wild type. Mutations that produced an antimutator phenotype were distributed throughout the polymerase domain, with 12% clustered in the M-helix. We confirmed that a single mutation in this segment results in increased base discrimination. Thus, this work identifies the M-helix as a determinant of fidelity and suggests that polymerases can tolerate many substitutions that alter fidelity without incurring major changes in activity.     

Contrasting effects of Escherichia coli single-stranded DNA binding protein on synthesis by T7 DNA polymerase and Escherichia coli DNA polymerase I (large fragment). Evidence that binding protein inhibits trans-lesion synthesis by polymerase I

The effect of Escherichia coli single-stranded DNA binding protein (SSB) on DNA synthesis by T7 DNA polymerase and E. coli DNA polymerase I (large fragment) using native or aminofluorene-modified M13 templates was evaluated by in vitro DNA synthesis assays and polyacrylamide gel electrophoresis analysis. The two polymerase enzymes displayed differential responses to the addition of SSB. T7 DNA polymerase, a enzyme required for the replication of the T7 chromosome, was stimulated by the addition of SSB whether native or modified templates were used. On the other hand, E. coli DNA polymerase I was slightly stimulated by the addition of SSB to the native template but substantially inhibited on modified templates. This result suggests that DNA polymerase I may be able to synthesize past an aminofluorene adduct but that the presence of SSB inhibited this trans-lesion synthesis. Polyacrylamide gels of the products of DNA synthesis by polymerase I supported this inference since SSB caused a substantial increase in the accumulation of shorter DNA chains induced by blockage at the aminofluorene adduct sites.     

Base-pairing properties of N4-methoxydeoxycytidine 5'-triphosphate during DNA synthesis on natural templates, catalyzed by DNA polymerase I of Escherichia coli

N4-Methoxydeoxycytidine 5'-triphosphate (mo4dCTP) was synthesized by reaction of dCTP with methoxyamine and then purified by high-performance liquid chromatography (HPLC) and used to analyze the specificity of mo4dCMP incorporation during polymerization on natural templates, catalyzed by DNA polymerase I of Escherichia coli. Elongation of synthetic 5'-32P-labeled primers, annealed to single-stranded DNA of bacteriophage M13, was carried out in the presence of only three of the four normal dNTPs; then, reaction products were displayed by high-resolution gel electrophoresis and visualized by autoradiography. By measuring primer elongation in each of the four "minus" reactions with and without added mo4dCTP, we examined the specificity of mo4dCMP incorporation at different positions along the M13 template. The results of this experimental approach indicated that (i) mo4dCTP is utilized most readily (although at low efficiency) in place of dTTP during DNA synthesis, (ii) the analogue can also replace dCTP during primer elongation, although at barely detectable efficiency, and (iii) the ease at which both mo4C.A and mo4C.G pairs are formed during DNA synthesis on natural templates is markedly influenced by the nucleotide sequence of the template.     

Translesional synthesis on a DNA template containing N2-methyl-2'-deoxyguanosine catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I

Formaldehyde is produced in most living systems and is present in the environment. Evidence that formaldehyde causes cancer in experimental animals infers that it may be a carcinogenic hazard to humans. Formaldehyde reacts with the exocyclic amino group of deoxyguanosine, resulting in the formation of N²-methyl-2′-deoxyguanosine (N²-Me-dG) via reduction of the Schiff base. The same reaction is likely to occur in living cells, because cells contain endogenous reductants such as ascorbic acid and gluthathione. To explore the miscoding properties of formaldehyde-derived DNA adducts a site-specifically modified oligodeoxynucleotide containing a N²-Me-dG was prepared and used as the template in primer extension reactions catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I. The primer extension reaction was slightly stalled one base before the N²-Me-dG lesion, but DNA synthesis past this lesion was readily completed. The fully extended products were analyzed to quantify the miscoding specificities of N²-Me-dG. Preferential incorporation of dCMP, the correct base, opposite the lesion was observed, along with small amounts of misincorporation of dTMP (9.4%). No deletions were detected. Steady-state kinetic studies indicated that the frequency of nucleotide insertion for dTMP was only 1.2 times lower than for dCMP and the frequency of chain extension from the 3′-terminus of a dT:N²-Me-dG pair was only 2.1 times lower than from a dC:N²-Me-dG pair. We conclude that N²-Me-dG is a miscoding lesion capable of generating G→A transition mutations.     

On the fidelity of DNA replication. The accuracy of Escherichia coli DNA polymerase I in copying natural DNA in vitro

The accuracy with which Escherichia coli DNA polymerase I (Pol I) copies natural DNA in vitro has been determined. When phi X174 viral DNA containing an amber mutation (am3) is primed with a single restriction endonuclease fragment, copied in vitro with Pol I and then expressed in E. coli spheroplasts (Weymout, L. A., and Loeb, L. A. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 1924), the reversion frequency of this DNA is greater than that of uncopied DNA. This change in reversion frequency can be increased by selectively increasing the concentration of either dATP or dCTP relative to the other deoxyribonucleotide substrates. DNA sequence analyses of revertants obtained from substrate pool bias experiments demonstrates that the revertants contain the selectively biased nucleotide as an incorrect substitution at position 587 of the am3 codon. We have analyzed the product of the in vitro Pol I reaction using neutral and alkaline sucrose gradients. Fifty per cent of the input phi X174 DNA template molecules are copied past the am3 site. The phenotypic expression of the product (revertant) strand in the spheroplast assay was estimated using a model heteroduplex molecule similar in structure to the product of the reaction and containing a single base mismatch (A:A or A:C) at position 587. Using these data, and by extrapolation from pool bias experiments, we estimate the error rate of Pol I in Mg2+-activated reactions using equimolar concentrations of the four deoxynucleotide substrates is 1/680,000 for an A:C mispair and < 1/6,300,000 for an A:A mispair at position 587 of the am3 codon in phi X174 DNA.     

DNA replication fidelity in Escherichia coli: a multi-DNA polymerase affair

High accuracy (fidelity) of DNA replication is important for cells to preserve the genetic identity and to prevent the accumulation of deleterious mutations. The error rate during DNA replication is as low as 10(-9) to 10(-11) errors per base pair. How this low level is achieved is an issue of major interest. This review is concerned with the mechanisms underlying the fidelity of the chromosomal replication in the model system Escherichia coli by DNA polymerase III holoenzyme, with further emphasis on participation of the other, accessory DNA polymerases, of which E.?coli contains four (Pols I, II, IV, and V). Detailed genetic analysis of mutation rates revealed that (1) Pol II has an important role as a back-up proofreader for Pol III, (2) Pols IV and V do not normally contribute significantly to replication fidelity, but can readily do so under conditions of elevated expression, (3) participation of Pols IV and V, in contrast to that of Pol II, is specific to the lagging strand, and (4) Pol I also makes a lagging-strand-specific fidelity contribution, limited, however, to the faithful filling of the Okazaki fragment gaps. The fidelity role of the Pol III τ subunit is also reviewed.     

Mechanisms of error discrimination by Escherichia coli DNA polymerase I

The mechanism of base selection by DNA polymerase I of Escherichia coli has been investigated by kinetic analysis. The apparent KM for the insertion of the complementary nucleotide dATP into the hook polymer poly(dT)-oligo(dA) was found to be 6-fold lower than that for the noncomplementary nucleotide dGTP, whereas the Vmax for insertion of dATP was 1600-fold higher than that for dGTP. The ratio of Kcat/KM values for complementary and mismatched nucleotides of 10(4) demonstrates the extremely high specificity of base selection by DNA polymerase I and is in agreement with results obtained with a different template-primer, poly(dC)-oligo(dG) [El-Deiry, W. S., Downey, K. M., & So, A. G. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 7378]. Studies on the effects of phosphate ion on the polymerase and 3'- to 5'-exonuclease activities of DNA polymerase I showed that, whereas the polymerase activity was somewhat stimulated by phosphate, the exonuclease activity was markedly inhibited, being 50% inhibited at 25 mM phosphate and greater than 90% inhibited at 80 mM phosphate. Selective inhibition of the exonuclease activity by phosphate also resulted in inhibition of template-dependent conversion of a noncomplementary dNTP to dNMP and, consequently, markedly affected the kinetic constants for insertion of noncomplementary nucleotides. The mutagenic metal ion Mn2+ was found to affect error discrimination by both the polymerase and 3'- and 5'-exonuclease activities of DNA polymerase I.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA strand transfer catalyzed by the 5'-3' exonuclease domain of Escherichia coli DNA polymerase I.

A protein which promotes DNA strand transfer between linear double-stranded M13mp19 DNA and single-stranded viral M13mp19 DNA has been isolated from recA- E.coli. The protein is DNA polymerase I. Strand transfer activity residues in the small fragment encoding the 5'-3' exonuclease and can be detected using a recombinant protein comprising the first 324 amino acids encoded by polA. Either the recombinant 5'-3' exonuclease or intact DNA polymerase I can catalyze joint molecule formation, in reactions requiring only Mg2+ and homologous DNA substrates. Both kinds of reactions are unaffected by added ATP. Electron microscopy shows that the joint molecules formed in these reactions bear displaced single strands and therefore this reaction is not simply promoted by annealing of exonuclease-gapped molecules. The pairing reaction is also polar and displaces the 5'-end of the non-complementary strand, extending the heteroduplex joint in a 5'-3' direction relative to the displaced strand. Thus strand transfer occurs with the same polarity as nick translation. These results show that E.coli, like many eukaryotes, possesses a protein which can promote ATP-independent strand-transfer reactions and raises questions concerning the possible biological role of this function.     

Role of Escherichia coli DNA polymerase IV in in vivo replication fidelity

We have investigated whether DNA polymerase IV (Pol IV; the dinB gene product) contributes to the error rate of chromosomal DNA replication in Escherichia coli. We compared mutation frequencies in mismatch repair-defective strains that were either dinB positive or dinB deficient, using a series of mutational markers, including lac targets in both orientations on the chromosome. Virtually no contribution of Pol IV to the chromosomal mutation rate was observed. On the other hand, a significant effect of dinB was observed for reversion of a lac allele when the lac gene resided on an F'(pro-lac) episome.     

Inducible DNA polymerase I synthesis in a UV hyper-resistant mutant of Escherichia coli

A mutant of Escherichia coli which is more resistant to shortwave UV light than its wild-type parent strain and which can synthesise DNA polymerase I constitutively has been further analysed. It carries two mutational alleles which are located about 1.5 min apart and cotransducible by P1 with the argH locus. The two mutational alleles have been segregated and their analysis shows that one of them is responsible for UV hyper-resistance whereas the other mutation confers UV sensitivity. Recombinant plasmids carrying various sections of the polA regulatory region, linked to a galK gene, were introduced into the mutant strains. Analysis of galactokinase shows that the enzyme activity in the UV hyper-resistant mutant is increased. The results suggest that the synthesis of DNA polymerase I in E. coli is inducible.