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• W2079355585 abstract "The fidelity of DNA replication byEscherichia coli DNA polymerase I (pol I) was assessedin vivo using a reporter plasmid bearing a ColE1-type origin and an ochre codon in the β-lactamase gene. We screened 53 single mutants within the region Val700–Arg712in the polymerase active-site motif A. Only replacement of Ile709 yielded mutator polymerases, with substitution of Met, Asn, Phe, or Ala increasing the β-lactamase reversion frequency 5–23-fold. Steady-state kinetic analysis of the I709F polymerase revealed reductions in apparent K m values for both insertion of non-complementary nucleotides and extension of mispaired primer termini. Abolishment of the 3′–5′ exonuclease activity of wild-type pol I increased mutation frequency 4-fold, whereas the combination of I709F and lack of the 3′–5′ exonuclease yielded a 400-fold increase. We conclude that accurate discrimination of the incoming nucleotide at the polymerase domain is more critical than exonucleolytic proofreading for the fidelity of pol I in vivo. Surprisingly, the I709F polymerase enhanced mutagenesis in chromosomal DNA, although the increase was 10-fold less than in plasmid DNA. Our findings indicate the feasibility of obtaining desired mutations by replicating a target gene at a specific locus in a plasmid under continuous selection pressure. The fidelity of DNA replication byEscherichia coli DNA polymerase I (pol I) was assessedin vivo using a reporter plasmid bearing a ColE1-type origin and an ochre codon in the β-lactamase gene. We screened 53 single mutants within the region Val700–Arg712in the polymerase active-site motif A. Only replacement of Ile709 yielded mutator polymerases, with substitution of Met, Asn, Phe, or Ala increasing the β-lactamase reversion frequency 5–23-fold. Steady-state kinetic analysis of the I709F polymerase revealed reductions in apparent K m values for both insertion of non-complementary nucleotides and extension of mispaired primer termini. Abolishment of the 3′–5′ exonuclease activity of wild-type pol I increased mutation frequency 4-fold, whereas the combination of I709F and lack of the 3′–5′ exonuclease yielded a 400-fold increase. We conclude that accurate discrimination of the incoming nucleotide at the polymerase domain is more critical than exonucleolytic proofreading for the fidelity of pol I in vivo. Surprisingly, the I709F polymerase enhanced mutagenesis in chromosomal DNA, although the increase was 10-fold less than in plasmid DNA. Our findings indicate the feasibility of obtaining desired mutations by replicating a target gene at a specific locus in a plasmid under continuous selection pressure. DNA polymerase I exonuclease-deficient 3′–5′ exonuclease-deficient DNA polymerase III DNA polymerases catalyze chain elongation reactions guided by complementary base pairings opposite a single-stranded DNA template. These reactions are highly accurate, exhibiting error rates of about one base substitution error per 104 to 107nucleotides polymerized (1Kunkel T.A. Bebenek K. Annu. Rev. Biochem. 2000; 69: 497-529Crossref PubMed Scopus (790) Google Scholar). However, errors made by the polymerase, if not subsequently excised, can become fixed as mutations during subsequent rounds of replication. As a result, errors by DNA polymerases can be a major source of spontaneous mutagenesis and can contribute to the multiplicity of mutations found in cancer cells (2Loeb L.A. Cancer Res. 2001; 61: 3230-3239PubMed Google Scholar,3Loeb K.R. Loeb L.A. Carcinogenesis. 2000; 21: 379-385Crossref PubMed Scopus (322) Google Scholar). Many DNA polymerases have intrinsic or associated 3′–5′ exonucleases that preferentially hydrolyze non-complementary nucleotides immediately after formation of the phosphodiester bond and contribute from a few- to 100-fold to the fidelity of DNA synthesis (4Brutlag D. Kornberg A. J. Biol. Chem. 1972; 247: 241-248Abstract Full Text PDF PubMed Google Scholar, 5Muzyczka N. Poland R.L. Bessman M.J. J. Biol. Chem. 1972; 247: 7116-7122Abstract Full Text PDF PubMed Google Scholar, 6Echols H. Lu C. Burgers P.M. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2189-2192Crossref PubMed Scopus (110) Google Scholar). In addition, errors introduced by DNA polymerases are subsequently corrected by a mismatch repair system, which contributes an additional 2–3 orders of magnitude to the accuracy of DNA replication (7Modrich P. J. Biol. Chem. 1989; 264: 6597-6600Abstract Full Text PDF PubMed Google Scholar). However, base selection at the polymerase active site during both the nucleotide insertion and subsequent extension reactions, including Watson-Crick base pair formation between complementary bases and a conformational change of the active site during each incorporation step, is likely the most significant contributor to the fidelity of DNA polymerization (1Kunkel T.A. Bebenek K. Annu. Rev. Biochem. 2000; 69: 497-529Crossref PubMed Scopus (790) Google Scholar, 8Pham P.T. Olson M.W. McHenry C.S. Schaaper R.M. J. Biol. Chem. 1998; 273: 23575-23584Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 9Bebenek K. Joyce C.M. Fitzgerald M.P. Kunkel T.A. J. Biol. Chem. 1990; 265: 13878-13887Abstract Full Text PDF PubMed Google Scholar, 10Kunkel T.A. J. Biol. Chem. 1992; 267: 18251-18254Abstract Full Text PDF PubMed Google Scholar). We have investigated the relationships between structure and function at the active site of DNA polymerases by substituting random sequences for nucleotides that encode residues at the active site and monitoring the effects of these substitutions on the fidelity of DNA synthesis (11Suzuki M. Avicola A.K. Hood L. Loeb L.A. J. Biol. Chem. 1997; 272: 11228-11235Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar,12Patel P.H. Kawate H. Adman E. Ashbach M. Loeb L.A. J. Biol. Chem. 2001; 276: 5044-5051Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Escherichia coli DNA polymerase I (pol I)1 is involved in DNA replication, DNA repair, and genetic recombination (13Kornberg A. Baker T. DNA Replication. 2nd Ed. W. H. Freeman & Co., New York1992Google Scholar); it is the most extensively studied of all DNA polymerases. Evidence indicates that pol I functions in DNA replication by removal of RNA primers and resynthesis of the resulting gaps between Okazaki fragments on the lagging strand (14Kuempel P.L. Veomett G.E. Biochem. Biophys. Res. Commun. 1970; 41: 973-980Crossref PubMed Scopus (57) Google Scholar, 15Okazaki R. Arisawa M. Sugino A. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 2954-2957Crossref PubMed Scopus (103) Google Scholar). In addition, pol I participates in DNA repair by filling gaps resulting from the excision of damaged bases (16Boyle J.M. Paterson M.C. Setlow R.B. Nature. 1970; 226: 708-710Crossref PubMed Scopus (52) Google Scholar, 17Cooper P.K. Hanawalt P.C. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 1156-1160Crossref PubMed Scopus (98) Google Scholar). Moreover, pol I is required for the initiation of synthesis at the origin of replication in certain plasmids (13Kornberg A. Baker T. DNA Replication. 2nd Ed. W. H. Freeman & Co., New York1992Google Scholar, 18del Solar G. Giraldo R. Ruiz-Echevarrı́a M.J. Espinosa M. Dı́az-Orejas R. Microbiol. Mol. Biol. Rev. 1998; 62: 434-464Crossref PubMed Google Scholar). The crystal structure of the Klenow fragment of pol I (which lacks the 5′–3′ exonuclease domain) reveals an architecture that is common among DNA polymerases and has been likened to a human right hand, with a fingers subdomain (which binds the incoming dNTP and interacts with the single-stranded DNA template), a thumb subdomain (which binds double-stranded DNA), and a palm subdomain (which harbors the catalytic amino acids and also interacts with the incoming dNTP) (19Ollis D.L. Brick P. Hamlin R. Xuong N.G. Steitz T.A. Nature. 1985; 313: 762-766Crossref PubMed Scopus (736) Google Scholar, 20Steitz T.A. J. Biol. Chem. 1999; 274: 17395-17398Abstract Full Text Full Text PDF PubMed Scopus (691) Google Scholar). Several mutant forms of the Klenow fragment of pol I (in which single amino acid substitutions have been introduced into the active-site fingers or palm subdomain (21Carroll S.S. Cowart M. Benkovic S.J. Biochemistry. 1991; 30: 804-813Crossref PubMed Scopus (116) Google Scholar, 22Bell J.B. Eckert K.A. Joyce C.M. Kunkel T.A. J. Biol. Chem. 1997; 272: 7345-7351Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 23Minnick D.T. Bebenek K. Osheroff W.P. Turner Jr., R.M. Astatke M. Liu L. Kunkel T.A. Joyce C.M. J. Biol. Chem. 1999; 274: 3067-3075Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) or a large 24-amino acid segment in the thumb subdomain has been deleted (24Minnick D.T. Astatke M. Joyce C.M. Kunkel T.A. J. Biol. Chem. 1996; 271: 24954-24961Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar)) that exhibit altered fidelity in DNA synthesis in vitro have been investigated. Although many such mutant enzymes exhibit reduced fidelity in vitro, none has been shown to alter accuracyin vivo. In a recent study, we examined the mutability of motif A, extending from Val700 to Arg712, in the palm subdomain ofE. coli pol I using random mutagenesis and a genetic complementation system (25Shinkai A. Patel P.H. Loeb L.A. J. Biol. Chem. 2001; 276: 18836-18842Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). We established a library of 500,000 transfectants and sequenced 232 of 37,500 mutants that were active in the complementation assay. E. coli strains harboring the active mutants were fit to replicate repetitively, and the mutant polymerases, when purified, displayed 20–190% of the wild-type specific activity. Thus, motif A is highly mutable while preserving wild type-like DNA polymerase activity in vitro and in vivo. The ease of substitutability of motif A residues revealed in this work, yielding highly functional variants, stands in sharp contrast to the marked conservation of the motif A sequence observed among prokaryotic DNA polymerases (26Astatke M. Grindley N.D.F. Joyce C.M. J. Mol. Biol. 1998; 278: 147-165Crossref PubMed Scopus (99) Google Scholar, 27Patel P.H. Suzuki M. Adman E. Shinkai A. Loeb L.A. J. Mol. Biol. 2001; 308: 823-837Crossref PubMed Scopus (158) Google Scholar). Interestingly, we also found that certain substitutions of Ile709 permit more efficient utilization of rNTPs as substrates in vitro. In this study, we screened 53 mutations in motif A for infidelity of DNA synthesis in vivo and found that mutant enzymes harboring Ile709 substitutions exhibited less accurate DNA replication. The mutator phenotype was enhanced when the Ile709 substitutions were combined with deficiency of 3′–5′ exonucleolytic proofreading activity. In subsequent in vitro experiments, we determined that the I709F substitution increased both insertion of non-complementary nucleotides as well as extension from primers with mismatched 3′-OH termini. To our knowledge, this is the first analysis of the effects of mutation in the polymerase active site of E. coli pol I on the fidelity of DNA synthesis both in vitro and in cells. The wild-type and mutant E. coli pol I genes were inserted into pHSG576 (28Takeshita S. Sato M. Toba M. Masahashi W. Hashimoto-Gotoh T. Gene (Amst.). 1987; 61: 63-74Crossref PubMed Scopus (593) Google Scholar), placing them under the control of the lactose promoter. pHSG576 is low copy number plasmid that has a pol I-independent origin. To modify the gene, the wild-type pol I gene of E. coli DH5α was amplified by colony polymerase chain reaction and inserted into pHSG576 to create pECpol I as described previously (25Shinkai A. Patel P.H. Loeb L.A. J. Biol. Chem. 2001; 276: 18836-18842Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Site-directed mutagenesis was performed on pECpol I to introduce an A-to-C transversion at position 1271, thus changing Asp424 to Ala and inactivating the 3′–5′ exonuclease activity (29Derbyshire V. Freemont P.S. Sanderson M.R. Beese L. Friedman J.M. Joyce C.M. Steitz T.A. Science. 1988; 240: 199-201Crossref PubMed Scopus (301) Google Scholar), to construct pECpol I-3′exo−, which carries the 3′–5′ exonuclease-minus pol I gene. Plasmids pECI709M, pECI709N, pECI709F, and pECI709A, which carry Ile709 mutant pol I genes, were isolated from a mutant pol I library by genetic selection as described previously (25Shinkai A. Patel P.H. Loeb L.A. J. Biol. Chem. 2001; 276: 18836-18842Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Plasmids pECI709M-3′exo−, pECI709N-3′exo−, pECI709F-3′exo−, and pECI709A-3′exo− were constructed by substituting the 1.1-kb SacI-EcoRI fragment of pECpol I-3′exo− for the corresponding fragment of pECI709M, pECI709N, pECI709F, and pECI709A, respectively. The reporter plasmids for measuring the reversion frequency of the β-lactamase gene were constructed by modifying plasmid pGPS3 (New England Biolabs Inc., Beverly, MA), which contains a ColE1-type origin derived from pUC19. Site-directed mutagenesis was performed on pGPS3 to introduce a G-to-T transversion at position 76 of the β-lactamase gene, changing the codon GAA for Glu26 to the ochre codon TAA. The resulting plasmid was designated pLA2800. The mutant β-lactamase gene containing its own promoter was amplified by polymerase chain reaction with the synthetic oligonucleotides 5′-GCACCCGACATACATGTCCTATTTGTTTATT-3′ and 5′-AAACTTGGTCGGATCCTTACCAATGCTTAATC-3′ as primers and pLA2800 as a template, and the amplified fragment was cloned into pCRII (Invitrogen, Carlsbad, CA). The 1-kb AflIII-KpnI fragment containing the mutant β-lactamase gene was excised and cloned into the AflIII-KpnI site ∼60 bp distant from the origin of pGPS3ΔLA (see below) to create pLA230. Plasmid pGPS3ΔLA was constructed by replacing the 1.2-kbBglII-BglI fragment of pGPS3 with the synthetic oligonucleotides 5′-GATCTGATCGCCCTTC-3′ and 5′-GGGCGATCA-3′. A schematic representation of the reporter plasmids pLA230 and pLA2800 is shown in Fig. 1 A. A schematic representation of this assay is shown in Fig. 1 B. E. coli JS200 (recA718 polA12 uvrA155 trpE65 lon-11 sulA1) (30Sweasy J.B. Loeb L.A. J. Biol. Chem. 1992; 267: 1407-1410Abstract Full Text PDF PubMed Google Scholar, 31Witkin E.M. Boegner-Maniscalco V. J. Bacteriol. 1992; 174: 4166-4168Crossref PubMed Google Scholar) harboring pLA230 or pLA2800 was transformed with plasmids carrying wild-type or mutant pol I genes. The recombinant strains were cultured at 30 °C for 16 h in nutrient broth containing 50 μg/ml kanamycin, 12.5 μg/ml tetracycline, and 30 μg/ml chloramphenicol. A 0.01 volume of the pre-cultured broth was inoculated into fresh medium; cultured at 37 °C until an A 600 of ∼1.0 was attained; and then plated onto LB agar plates supplemented with 50 μg/ml kanamycin, 12.5 μg/ml tetracycline, and 30 μg/ml chloramphenicol in the presence or absence of 80 μg/ml carbenicillin. After incubation at 37 °C for 16 h, colonies were counted, and reversion frequencies were calculated as the ratio of carbenicillin-resistant to total colonies. Plasmids prepared from revertants were re-transformed into E. coli BL21 and selected on LB plates containing 50 μg/ml carbenicillin. The plasmids were prepared from the recombinant BL21 strain, and the nucleotide sequence of the entire β-lactamase gene was determined. The Trp+reversion assay was performed basically following the method of Washington et al. (32Washington S.L. Yoon M.S. Chagovetz A.M. Li S.-X. Clairmont C.A. Preston B.D. Eckert K.A. Sweasy J.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1321-1326Crossref PubMed Scopus (55) Google Scholar). E. coli JS200 was transformed with plasmids pECpol I, pECpol I-3′exo−, pECI709F, and pECI709F-3′exo−, as indicated. The transfectants were cultured at 30 °C for 16 h in nutrient broth supplemented with 30 μg/ml chloramphenicol and 12.5 μg/ml tetracycline. A 0.01 volume of each culture was inoculated into fresh medium, cultured at 37 °C until an A600 of ∼1.0 was attained, and then plated onto M9 minimum agar plates supplemented with 30 μg/ml chloramphenicol in the presence or absence of 40 μg/ml tryptophan. After incubation at 37 °C for 20 h, colonies were counted, and the frequency of appearance of the Trp+ strain was calculated. The trpE gene in JS200 and in Trp+revertant strains was amplified by colony polymerase chain reaction with 5′-CCATGCGTAAAGCAATCAGATACCC-3′ and 5′-TTATCGAGCAGCAGAATGTCAGCCA-3′ as primers, and the amplified fragment was cloned into pCRII; the nucleotide sequence of the entiretrpE gene was then determined. Steady-state kinetic analysis of misincorporation frequency was performed based on the method of Boosalis et al. (33Boosalis M.S. Petruska J. Goodman M.F. J. Biol. Chem. 1987; 262: 14689-14696Abstract Full Text PDF PubMed Google Scholar). A 47-mer template (3′-GCGCGGCTTAAGGGCGATCGTTATAGCTTAAGGCCTTTAAAGGGCCC-5′; the relevant template bases are underlined) was hybridized with a32P-5′-end-labeled 23-mer primer (5′-CGCGCCGAATTCCCGCTAGCAAT-3′) for analysis of misinsertion efficiency opposite dT and with a 25-mer primer (5′-CGCGCCGAATTCCCGCTAGCAATAT-3′) for analysis opposite dG. Primer-template (5 nm) was incubated for 5 min at 37 °C in a reaction mixture containing limiting amounts of purified recombinant Klenow (exo−) protein (5 nm) prepared as described previously (25Shinkai A. Patel P.H. Loeb L.A. J. Biol. Chem. 2001; 276: 18836-18842Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) and varying concentrations of each dNTP in 10 mm Tris-HCl (pH 7.5), 5 mm MgCl2, and 7.5 mmdithiothreitol. The ranges of nucleotide substrate concentrations used for measuring incorporation opposite template dT were 0.5–7.5 nm dATP, 0.5–50 μm dGTP, and 10–300 μm dCTP and dTTP for the wild-type enzyme and 0.5–7.5 nm dATP, 0.05–5 μm dGTP, 2–14 μm dCTP, and 1–7 μm dTTP for the I709F mutant enzyme. The concentrations of the nucleotide substrates opposite template dG were 10–70 μm dATP, 1–50 μmdGTP, 2–30 nm dCTP, and 10–300 μm dTTP for the wild-type enzyme and 0.1–5 μm dATP, 0.1–5 μm dGTP, 2–30 nm dCTP, and 0.1–30 μm dTTP for the I709F mutant enzyme. Following termination of the reaction by addition of 2.5 μl of formamide solution, the products were analyzed by 14% polyacrylamide gel electrophoresis and quantified by phosphor image analysis (34Patel P.H. Loeb L.A. J. Biol. Chem. 2000; 275: 40266-40272Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Mismatch extension frequency was determined using a similar protocol, except that the sequence of the 24-mer primer was 5′-CGCGCCGAATTCCCGCTAGCAATX-3′ (where X was A, G, C, or T). Reaction mixtures contained dTTP, i.e. the correct dNTP for insertion opposite the next template base. The efficiency of dTTP incorporation opposite template dA was measured for each primer-template construct. The concentrations of the dTTP substrate were 0.5–3.5 nm for the T:A matched pair, 5–1000 nm for the T:G and T:C mismatches, and 0.025–14 μm for the T:T mismatch. To measure errors in DNA synthesis by pol I in vivo, we established a two-plasmid system. The reporter plasmid pLA230 (Fig. 1 A) contains a β-lactamase gene harboring an ochre mutation near the 5′ terminus. Since evidence indicates that pol I is involved in initiation of DNA synthesis in ColE1-type plasmids (13Kornberg A. Baker T. DNA Replication. 2nd Ed. W. H. Freeman & Co., New York1992Google Scholar, 18del Solar G. Giraldo R. Ruiz-Echevarrı́a M.J. Espinosa M. Dı́az-Orejas R. Microbiol. Mol. Biol. Rev. 1998; 62: 434-464Crossref PubMed Google Scholar), we introduced the ochre mutation ∼230 bp from the ori sequence. The reporter plasmid, together with a second plasmid encoding the wild-type or mutant pol I gene, was transfected into E. coli JS200 (30Sweasy J.B. Loeb L.A. J. Biol. Chem. 1992; 267: 1407-1410Abstract Full Text PDF PubMed Google Scholar, 31Witkin E.M. Boegner-Maniscalco V. J. Bacteriol. 1992; 174: 4166-4168Crossref PubMed Google Scholar), a strain that contains a temperature-sensitive pol I. The reversion frequency at the β-lactamase locus was determined by measuring colony formation in the presence and absence of carbenicillin (Fig. 1 B). The pol I gene encoded by the second plasmid corresponds to the intact enzyme and hence encodes both 5′–3′ and 3′–5′ exonuclease activities as well as DNA polymerase activity (13Kornberg A. Baker T. DNA Replication. 2nd Ed. W. H. Freeman & Co., New York1992Google Scholar). To identify amino acids in active-site motif A that affect the fidelity of DNA synthesis, we tested 53 different single motif A mutations within the segment spanning Val700–Arg712 (25Shinkai A. Patel P.H. Loeb L.A. J. Biol. Chem. 2001; 276: 18836-18842Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Representative reversion frequencies obtained for mutants containing substitutions at each of the positions analyzed are shown in Table I. The reversion frequency observed for wild-type pol I was ∼1 × 10−7, as was that for all the mutants tested except the four with substitutions at position 709. Substitution of Met, Asn, Phe, or Ala for Ile709 yielded 5.3–23 times higher reversion frequencies than that for wild-type pol I (Tables I and II). We analyzed the nucleotide sequence of the β-lactamase reporter gene from five independent revertants harboring I709F mutant pol I and confirmed that the ochre mutation was converted twice to TTA, twice to TCA, and once to CAA. The enhanced mutagenesis observed for the Ile709 mutants provides new evidence thatE. coli DNA polymerase I is involved in plasmid replication by copying nucleotides near the ori sequence and demonstrates that Ile709 is critical for accurate plasmid replication in vivo.Table IReversion frequency at an ochre codon in the β-lactamase gene in E. coli expressing wild-type and mutant pol Ipol I1-aThe polymerases listed exhibited the highest reversion frequencies observed among mutants at each position in motif A.No. of colonies × 10−8/ml (−carbenicillin)No. of colonies/ml (+carbenicillin)Reversion frequency × 107Wild-type8.7901.0V700I101401.4I701N9.41201.3V702A9.4700.7S703R8.5901.1A704S8.81101.3Asp7051-bAsp705 is immutable (25).Y706F9.0600.7S707A8.6700.8Q708H9.2800.9I709N9.1210023E710D8.6300.3L711V8.91601.8R712S9.6800.81-a The polymerases listed exhibited the highest reversion frequencies observed among mutants at each position in motif A.1-b Asp705 is immutable (25Shinkai A. Patel P.H. Loeb L.A. J. Biol. Chem. 2001; 276: 18836-18842Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Open table in a new tab To analyze the contribution of the 3′–5′ exonuclease (“proofreading”) activity of pol I to the fidelity of plasmid replication, we mutated the 3′–5′ exonuclease in the wild-type enzyme and in the Ile709 variants by substituting Ala for Asp at position 424 in the exonuclease domain. When introduced into the wild-type construct, the D424A substitution enhanced the reversion frequency of the β-lactamase gene by 4.4-fold (Table II). Larger increases (up to 22-fold) were observed for specific amino acid substitutions of Ile709, indicating that discrimination at the active site can be more important for fidelity than exonucleolytic proofreading. Abolishment of the 3′-exonuclease activity in the mutants harboring the I709M, I709N, I709F, or I709A substitution resulted in a 29–416-fold increase in mutation frequency relative to the wild-type enzyme. For each of the mutants, the increase in reversion frequency associated with inactivation of the exonuclease was greater than that observed for the wild-type enzyme. In the case of I709F, the increase was substantially greater than multiplicative, suggesting a functional interaction between the exonuclease domain and motif A.Table IIReversion frequency of the β-lactamase gene in E. coli expressing wild-type and mutant DNA pol I lacking 3′–5′ exonuclease activitypol IReversion frequency2-aValues represent means ± S.D. obtained by plating three independent clones.Relative frequencyWild-type(1.2 ± 0.7) × 10−713′ exo−(5.3 ± 1.6) × 10−74.4I709M(6.3 ± 3.2) × 10−75.3I709M/3′ exo−(3.5 ± 0.3) × 10−629I709N(2.6 ± 0.6) × 10−622I709N/3′ exo−(3.3 ± 1.5) × 10−5280I709F(1.6 ± 0.1) × 10−613I709F/3′ exo−(5.0 ± 0.1) × 10−5416I709A(1.5 ± 0.2) × 10−613I709A/3′ exo−(1.2 ± 0.1) × 10−51002-a Values represent means ± S.D. obtained by plating three independent clones. Open table in a new tab To further evaluate the mutant polymerases, we measured the reversion frequency of the same β-lactamase gene on another plasmid, pLA2800 (Fig. 1 A and Table III). In this construct, the β-lactamase gene is located ∼2.8 kb downstream of the origin of replication and thus is ∼10-fold more distant from the origin than in pLA230. Introduction of the 3′exo−mutation into the wild-type construct or substitution of Met, Asn, Phe, or Ala for Ile709 in separate constructs resulted in at most a 1.8-fold increase in reversion frequency. In contrast, pol I harboring both the 3′exo− mutation and an Ile709 substitution showed 10–87-fold higher reversion frequency than wild-type pol I; the elevations were not as large, however, as those observed for pLA230 (Table II and III). These results suggest that DNA synthesis by pol I is not necessarily limited to nucleotides near the origin, but can occur much farther downstream.Table IIIReversion frequency of the β-lactamase gene located distally from the origin of plasmid replicationpol IReversion frequency3-aValues represent means ± S.D. of three determinations.Relative frequencyWild-type(1.5 ± 0.4) × 10−713′ exo−(1.3 ± 0.9) × 10−70.9I709M(1.0 ± 0.1) × 10−70.7I709M/3′ exo−(1.0 ± 0.5) × 10−66.7I709N(2.7 ± 1.1) × 10−71.8I709N/3′ exo−(5.2 ± 0.9) × 10−635I709F(1.4 ± 0.6) × 10−70.9I709F/3′ exo−(1.3 ± 0.4) × 10−587I709A(1.9 ± 0.7) × 10−71.3I709A/3′ exo−(2.9 ± 1.0) × 10−6193-a Values represent means ± S.D. of three determinations. Open table in a new tab E. coli JS200 cannot grow in the absence of tryptophan since it carries the trpE65 (ochre) allele in the host chromosome. We investigated the effects of the mutant polymerases on the reversion frequency at the trpE locus (Table IV). The reversion frequency observed for wild-type pol I was 2.0 × 10−8. Neither the 3′exo− mutation nor the I709F substitution significantly increased this frequency. In contrast, the mutant pol I with both the 3′exo− mutation and the I709F substitution exhibited a 40-fold increase in reversion frequency. We analyzed the nucleotide sequence of the trpE gene from three independent revertants and determined that the ochre mutation was converted once to TAC and twice to TCA. These results indicate that the mutant pol I bearing both an Ile709 substitution and the 3′exo− mutation participates in replication of theE. coli genome with less accuracy than the wild-type enzyme. However, the effect of the mutator activity on chromosomal DNA synthesis was less than on plasmid DNA synthesis. In all of thein vivo situations examined, specific mutations at the polymerase active site and inactivation of the proofreading activity acted synergistically to increase the mutator activity of pol I.Table IVReversion frequency at an ochre codon in the chromosomal trpE65 gene in E. coli expressing wild-type and mutant pol Ipol IReversion frequency4-aValues represent means ± S.D. obtained by analyzing three clones.Relative frequencyWild-type(2.0 ± 0.8) × 10−813′ exo−(3.0 ± 2.2) × 10−81.5I709F(2.3 ± 1.3) × 10−81.2I709F/3′ exo−(0.8 ± 0.2) × 10−6404-a Values represent means ± S.D. obtained by analyzing three clones. Open table in a new tab To establishin vitro correlates of the mutator phenotype of the Ile709 mutants, we purified the Klenow fragments of the wild-type and I709F exo− polymerases to apparent homogeneity (25Shinkai A. Patel P.H. Loeb L.A. J. Biol. Chem. 2001; 276: 18836-18842Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). These fragments lack both 5′–3′ and 3′–5′ exonuclease activities. The 5′–3′ exonuclease could remove the 5′-label from the primer, and the 3′–5′ exonuclease could remove added nucleotides in extension experiments. We then analyzed the efficiency of misinsertion using a steady-state gel-based assay (33Boosalis M.S. Petruska J. Goodman M.F. J. Biol. Chem. 1987; 262: 14689-14696Abstract Full Text PDF PubMed Google Scholar) to measure the kinetics of single nucleotide addition opposite template dT or dG. The primer was a 23- or 25-nucleotide oligomer that was labeled at the 5′-end with 32P, and the 3′-terminal nucleotide was one residue upstream from the target. The wild-type and mutant enzymes showed typical Michaelis-Menten saturation kinetics when initial velocity was plotted against the concentration of each nucleotide (data not shown). Apparent kinetic parameters and relative insertion frequencies were determined for each dNTP (Table V). The I709F polymerase incorporated complementary nucleotides with a catalytic efficiency indistinguishable from that of the wild-type enzyme. However, the catalytic efficiency of misincorporation of the non-complementary nucleotides was 6–35 times greater than that of the wild-type enzyme; the enhancement was 6–23-fold for misinsertion opposite template T and 8–35-fold opposite template G. Increased misincorporation by the mutant enzyme was due almost exclusively to a lower K m for mispaired dNTPs. Notably, misincorporation opposite dT parallels our in vivo finding of A-to-C or A-to-T transversions among the plasmid-borne β-lactamase revertants. Based on current models of initiation at ColE1-type origins (13Kornberg A. Baker T. DNA Replication. 2nd Ed. W. H. Freeman & Co., New York1992Google Scholar, 18del Solar G. Giraldo R" @default.
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• W2079355585 title "In Vivo Mutagenesis by Escherichia coliDNA Polymerase I" @default.
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