O-helix Mutant T664P of Thermus aquaticus DNA Polymerase I

Abstract

Previous studies indicate that the O-helix ofThermus aquaticus (Taq) DNA polymerase I (pol I) plays an important role in the replication fidelity of the enzyme. This study examines the role of Thr-664, which lies in the middle of the O-helix of Taq pol I. A mutant ofTaq Pol I with a proline substitution of Thr-664 (T664P) exhibits much lower replication fidelity than the wild type enzyme in a forward mutation assay. T664P produces base substitution, single-base deletion, and single-base insertion errors at 20-, 5, and 50-fold higher rates than wild type, respectively. In specific activity and steady-state kinetic experiments, T664P was catalytically robust for insertion of correct nucleotides. In contrast, it incorporated incorrect nucleotides 6.1- to 10-fold more efficiently than wild type at a template dC. Mismatched primer termini were extended by T664P 4.2- to 9.5-fold more efficiently than wild type. These data imply that the O-helix with a proline at position 664 functions like wild type Taq pol I for correct nucleotide incorporations, but bends and enlarges the catalytic pocket of the enzyme and increases the rate of nucleotide misincorporation. Previous studies indicate that the O-helix ofThermus aquaticus (Taq) DNA polymerase I (pol I) plays an important role in the replication fidelity of the enzyme. This study examines the role of Thr-664, which lies in the middle of the O-helix of Taq pol I. A mutant ofTaq Pol I with a proline substitution of Thr-664 (T664P) exhibits much lower replication fidelity than the wild type enzyme in a forward mutation assay. T664P produces base substitution, single-base deletion, and single-base insertion errors at 20-, 5, and 50-fold higher rates than wild type, respectively. In specific activity and steady-state kinetic experiments, T664P was catalytically robust for insertion of correct nucleotides. In contrast, it incorporated incorrect nucleotides 6.1- to 10-fold more efficiently than wild type at a template dC. Mismatched primer termini were extended by T664P 4.2- to 9.5-fold more efficiently than wild type. These data imply that the O-helix with a proline at position 664 functions like wild type Taq pol I for correct nucleotide incorporations, but bends and enlarges the catalytic pocket of the enzyme and increases the rate of nucleotide misincorporation. Thermus aquaticus polymerase Chromosomal DNA is subject to damage from naturally occurringin vivo processes such as hydrolysis, oxidation, and methylation (1Lindahl T. Nature. 1993; 362: 709-715Crossref PubMed Scopus (4342) Google Scholar). Cells have several DNA repair pathways that act as mutation avoidance systems and prevent the potentially deleterious effects of DNA damage. In addition to these systems, DNA polymerases discriminate between correct and incorrect nucleotides during DNA synthesis, thus providing accurate DNA synthesis and enhancing genomic stability (2Kunkel T.A. Bebenek K. Annu. Rev. Biochem. 2000; 69: 497-529Crossref PubMed Scopus (799) Google Scholar). During DNA synthesis by a polymerase, both the efficiency and the fidelity catalysis depends on the same phosphoryl transfer reaction and involves functionally and physically overlapping regions of the polymerase active site. The characteristics of mutant DNA polymerases are often useful for understanding the mechanisms that determine DNA replication fidelity. Among the mutants, we are interested in mutants with reduced fidelity but normal levels of catalytic activity. These types of mutations might be advantageous to a cell under conditions when low replication fidelity is desirable (i.e. to bypass DNA lesions) and could have played a role during evolution of error-prone DNA polymerases. Nucleotide selection fidelity could be altered by mutations that modify the interaction between polymerase and substrate/template-primer DNA (2Kunkel T.A. Bebenek K. Annu. Rev. Biochem. 2000; 69: 497-529Crossref PubMed Scopus (799) Google Scholar). For example, ribonucleotides are excluded from the DNA polymerase active site by structural/steric interference involving the 2′-OH group of the ribose sugar (3Gao G. Orlova M. Georgiadis M.M. Hendrickson W.A. Goff S.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 407-411Crossref PubMed Scopus (165) Google Scholar, 4Astatke M. Ng K. Grindley N.D. Joyce C.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3402-3407Crossref PubMed Scopus (182) Google Scholar, 5Patel P.H. Loeb L.A. J. Biol. Chem. 2000; 275: 40266-40272Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Mutations in Glu-615 inTaq 1 pol I, and the corresponding amino acid in Klenow or HIV reverse transcriptase, remove the steric clash with the 2′-OH group. Mutations in Ile-614 also reduce steric hindrance for incoming ribonucleotides and allow ribonucleotide misincorporation. In other mutant polymerases, an increase or decrease in the rate of base substitution or frameshift mutation is observed. Taq pol I F667L is a transversion antimutator in which the stacking force against bulky purine:purine base pairs may be reduced (6Suzuki M. Yoshida S. Adman E.T. Blank A. Loeb L.A. J. Biol. Chem. 2000; 275: 32728-32735Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). This mutant and most other polymerases with altered replication fidelity show altered catalytic efficiency; Taq pol I F667L demonstrates a 23-fold lower A→T transversion frequency and ∼30- to 100-fold lower catalytic efficiency than wild type Taq pol I. This work reports the properties of Taq pol I mutants that have substitutions at Thr-664 in the O-helix. Taq pol I T664P is a mutator polymerase whose catalytic properties are near wild type for insertion of correct nucleotides. It is proposed thatTaq pol I T664P may be a mutator because of increased plasticity in a wall of the catalytic pocket. Evidence supporting this hypothesis is discussed. Wild type and mutants in Taq pol I were purified as described previously (7Suzuki 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). Activity was measured in assays containing 200 µg/ml activated calf thymus DNA, 100 µm each dATP, dGTP, dCTP, and dTTP and 0.2 µCi of [3H]dATP in 50 mm Tris-HCl (pH 8.0), 2 mm MgCl2; incubation was for 5 min at 72 °C. Incorporation of radioactivity into an acid-insoluble product was determined according to Yoshida et al. (8Yoshida S. Kondo T. Ando T. Biochim. Biophys. Acta. 1974; 353: 463-474Crossref PubMed Scopus (98) Google Scholar). One unit represents incorporation of 10 nmol dNMP in 1 h, corresponding to 0.1 unit as defined by PerkinElmer Life Sciences (Foster City, CA). Oligonucleotides were synthesized and purified by Amersham Pharmacia Biotech (Buckinghamshire, UK). Replication error was quantitated by the M13mp2 forward mutation assay. A gapped M13mp2 substrate was constructed in which the single-stranded gap region contained thelacZα-complementation target sequence (9Bebenek K. Kunkel T.A. Methods Enzymol. 1995; 262: 217-232Crossref PubMed Scopus (192) Google Scholar). Gap-filling reactions were monitored by electrophoresis of the reaction products in 0.8% agarose gels. The reaction mixture contained 100 units of wild type or mutant Taq pol I, 400 ng of gapped M13mp2, 50 mm Tris-HCl (pH 8.0), 7 mm MgCl2, 50 mm KCl, and 200 µm each dNTP (6Suzuki M. Yoshida S. Adman E.T. Blank A. Loeb L.A. J. Biol. Chem. 2000; 275: 32728-32735Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). After incubation at 72 °C for 5 min, double-stranded phage DNA was transfected, the cultures were plated and incubated, and plaques were scored. Except for the experiment with T664P, mutant plaques were identified by their color. In some cases, plaques were isolated, replated and re-evaluated. DNA sequence was determined with an ABI PRISMTM Dye Primer Cycle Sequencing Core kit (Amersham Pharmacia Biotech, Buckinghamshire, UK). In earlier experiments, the number of background mutant plaques was as high as 3.5 × 10−3 (7Suzuki 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), making it difficult to estimate the increase or decrease in mutation frequencies for polymerases. However, the background decreased to 0.87 × 10−3 (6Suzuki M. Yoshida S. Adman E.T. Blank A. Loeb L.A. J. Biol. Chem. 2000; 275: 32728-32735Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), when a different preparation of gapped DNA substrate was used. In addition, the magnesium concentration was raised to 7 mm to force misincorporation (10Eckert K.A. Kunkel T.A. Nucleic Acids Res. 1990; 18: 3739-3744Crossref PubMed Scopus (312) Google Scholar). These changes in assay conditions made it possible to easily measure the mutation frequency of wild type polymerases and provided a sufficient number of mutant plaques for analysis. Deoxynucleotide concentrations were also increased to 200 µm to ensure efficient incorporation by polymerases with high Km values. The 14-mer DNA primer (5′-CGCGCCGAATTCCC) was 32P-labeled at the 5′-end by incubating with T4 polynucleotide kinase and [γ-32P]ATP and annealed to a 2-fold molar excess of the 46-mer DNA template, 5′- GCGCGGAAGCTTGGCTGCAGAATATTGCTAGCGGGAATTCGGCGCG. The labeled template-primer was mixed with unlabeled DNA in a 1:1 ratio and used at a final concentration of 50 nm in each reaction. Reaction mixtures contained 50 mm Tris-HCl (pH 8.0), 10 mm MgCl2, 50 mm KCl, 20 µm each dNTP, and 20 nm wild-type protein or 3.5 nm T664P in a final volume of 20 µl. In some experiments, either dCTP or dGTP was omitted from the mixture. Primer extension was carried at °C for 5, and nucleotide incorporation were the same as described previously S. J. Rev. Biochem. Biol. 1993; PubMed Scopus Google Scholar). 32P-labeled template-primer was mixed with unlabeled template-primer in a ratio and used at a final concentration of 50 nm in each reaction containing 50 mm Tris-HCl (pH 8.0), 10 50 mm KCl, and concentration of dNTP and enzyme as described measure incorporation of and and were that are to the of the 46-mer Incorporation of dNTP was monitored for kinetic under the conditions for which to of the template-primer was In reactions for correct nucleotide concentrations of wild-type protein and substrate were nm and µm dGTP, 2 nm and 2 nm and µm and nm and µm dCTP, respectively. In reactions with T664P, concentrations of the polymerase and substrate were nm and µm dGTP, 2 nm and µm dATP, 1 nm and µm and nm and µm dCTP, respectively. were at °C for 5 In reactions for incorrect nucleotide incorporation at template concentrations of wild-type protein and substrate were nm and µm dATP, µm and nm and µm were at °C for and min for incorporation of dATP, and dCTP, respectively. In reactions with T664P, concentrations of the polymerase and substrate were nm and µm dATP, 10 nm and µm and µm dCTP, respectively. were at °C for and min for incorporation of dATP, and dCTP, respectively. were by addition of an volume of 10 mm (pH 8.0), and by containing were for and the products were quantitated a Reaction efficiency was from the ratio of extended products and for the incorporation of each dNTP were from Primer extension efficiency for or base pairs was measured as described S. J. Rev. Biochem. Biol. 1993; PubMed Scopus Google Scholar, M.M. Nucleic Acids Res. PubMed Scopus Google Scholar). The of 32P-labeled template-primer was mixed with unlabeled template-primer in a ratio and used at a final concentration of 50 nm in each reaction containing 50 mm Tris-HCl (pH 8.0), 10 mm MgCl2, 50 mm KCl, and concentrations of dCTP and enzyme as described In some experiments, 5 nm template-primer was used with other conditions In concentrations of wild type protein and dCTP for template and were nm and 100 nm and nm and and nm and respectively. were carried for 5, and min for template and respectively. In reactions with T664P, concentrations of the polymerase and dCTP were nm and 5 nm and nm and and nm and respectively. were carried at °C for 5, and min for template and respectively. The incorporation of the dCTP was monitored for kinetic analysis. The were determined by the kinetic concentrations and 5 of template-primer S. M.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). In the of Thermus DNA polymerase I (pol I) that are by al. S. G. J. 1998; PubMed Scopus Google Scholar), we that substitution of Thr-664 with proline (T664P) at in the In wild pol I, the group of Thr-664 with the position of the base with the incoming 1 and a interaction between the Thr-664 group and the or amino group for other However, this interaction in the or S. G. Sci. 1998; PubMed Scopus Google Scholar). Thr-664 also a between the amino group and group These in the T664P study the functions of these we measured replication fidelity and kinetic of wild type and Thr-664 mutant polymerases. Replication fidelity was measured as the frequency of mutations during a of DNA a gapped M13mp2 DNA with the as template for DNA These assays were carried a of the previously 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 and the conditions wild type Taq pol I more mutant the mutation frequencies of high and low fidelity mutants are more easily measured and with the wild type enzyme (6Suzuki M. Yoshida S. Adman E.T. Blank A. Loeb L.A. J. Biol. Chem. 2000; 275: 32728-32735Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). In with wild type and Thr-664 mutants of Taq Pol I, the mutant frequency of wild type was × the frequency of × 10−3 is used in this which is the of several I of M. Yoshida S. Adman E.T. Blank A. Loeb L.A. J. Biol. Chem. 2000; 275: 32728-32735Abstract Full Text Full Text PDF PubMed Scopus (62) Google in The mutant frequency of and is to wild type The mutant frequency of and is higher than wild type, at and × respectively. The mutant frequency of was to that previously (7Suzuki 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), to wild type was lower because of the higher mutant frequency for wild frequencies for wild type Taq pol I in a forward mutation frequency of wild type and mutant Taq pol I mutant was determined by from color. frequency for this × was from each data For assays with Taq pol I T664P, plaques were to determine their from frequency of wild type and mutant Taq pol I mutant was determined by from color. frequency for this × was from each data For assays with Taq pol I T664P, plaques were to determine their from M. Yoshida S. Adman E.T. Blank A. Loeb L.A. J. Biol. Chem. 2000; 275: 32728-32735Abstract Full Text Full Text PDF PubMed Scopus (62) Google in a In most cases, mutant plaques are by and plaques on that have a much number of In were are isolated, mixed with wild type and plated in a However, it was difficult to the plaques by Taq Pol I T664P by this because the number of mutant plaques was and because the mutant plaques a of It was that at of the plaques were but the of the plaques was we the plaques as also of mutations and determined the DNA sequence for the plaques on the to determine the mutant frequency for this the mutant frequency of T664P was × 10−3 in the forward mutation assay The DNA sequence was determined for DNA from plaques during a forward mutation assay with were with mutation and with or more substitution and frameshift frequencies were × 10−3 error and × error respectively. These error frequencies are and higher than wild type for base substitution and frameshift of errors by wild type Taq pol I and T664P in a forward mutation from frequency was determined as described previously by and Kunkel The target region to This region for base substitution and for frameshift mutation frequency was determined by nucleotides in The number of mutations was by the number of nucleotides base from M. Yoshida S. Adman E.T. Blank A. Loeb L.A. J. Biol. Chem. 2000; 275: 32728-32735Abstract Full Text Full Text PDF PubMed Scopus (62) Google frequency was determined as described previously by and Kunkel M. Hood L. Loeb L.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The target region to This region for base substitution and for frameshift mutation (9Bebenek K. Kunkel T.A. Methods Enzymol. 1995; 262: 217-232Crossref PubMed Scopus (192) Google frequency was determined by nucleotides in The number of mutations was by the number of nucleotides in a The types of base substitutions and made by T664P are in substitution frequencies were in the of to higher than wild type. T664P produces and A→T at a high and these rates are and higher in the mutant than in wild type. In addition, and are made by T664P at a and higher rate than wild type. These data show that T664P between the correct and incorrect nucleotide and thus is a base substitution Taq pol I T664P is also a frameshift frequencies of base and were and higher than wild type, The of wild type and T664P was for the target region used in the forward mutation assay. The base substitutions made by T664P and the in contrast, the base substitutions made by wild type Taq pol I are in base substitutions in the with or sequence and are the target This is by number of base at base the substitution For and were at the of respectively. For and were at also mutation frequency for wild type and mutant of Taq pol I. For example, of base substitutions made by T664P are In the wild type of at the sequence However, in the of T664P, are more with or at the 5′- (i.e. were also with wild type and T664P a primer extension assay in the of dCTP or The and wild type or mutant enzyme were for to in wild type I the at template but efficiently the nucleotide because correct dCTP is of the reaction product However, by product at the position increases with incubation the product is efficiently to the nucleotide template at the primer at both the incorporation and extension also in the of dGTP of the primer as a much is extended to a and extension is because of the nucleotide template at the primer the of min, wild type Taq pol I of the template and of the template when dCTP or dGTP is omitted from the respectively. In contrast, Taq pol I T664P of the primer the template in the reaction dCTP, and of the primer the template in the reaction In addition to this high and extension reaction products target the dCTP the dGTP was incorporated by polymerase, a of is extended by Taq pol I T664P at These data indicate that T664P a than wild type to in the dCTP because of increased to and primer than because of a in the of activity in the reaction with mutant or wild type enzyme. steady-state reaction products were and the kinetic were determined the changes in T664P catalytic properties were to wild type. The insertion efficiency for dTTP by Taq pol I T664P was to wild type and was than wild type for dGTP, dATP, and dCTP The for and primer termini were also for wild type and T664P mutant Taq pol I. This is with the that the specific activity of T664P was of the wild type and that T664P the of M. Hood L. Loeb L.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google nucleotide insertion efficiency is a with of the correct dGTP as efficiency is a with of the correct dGTP as × × × × × × × × × × × × × × × × for insertion of correct incorrect nucleotides. The are the of efficiency is a with of the correct dGTP as in a for insertion of correct incorrect nucleotides. The are the of Wild type and T664P mutant Taq pol I polymerase have Km for misincorporation of and In contrast, were higher for the T664P × × 10−3 and × 10−3 × higher than wild pol I for and misincorporation respectively. a misincorporation by T664P were and 10 and higher than wild type. In the primer extension reaction that have nucleotides at the wild type and T664P also have for incorporation with a and a but T664P a and lower Km for and were also different for wild type and T664P Taq pol × 10−3 and × and × and × and × 10−3 For I T664P, extension of and were × × × × and × × higher than for wild type Taq pol nucleotide extension was determined according to efficiency is a with of the correct dGTP as efficiency is a with of the correct dGTP as × × × × × × × × × × × × × × × × × for extension of primer The are the of was determined according to M.M. Nucleic Acids Res. PubMed Scopus Google efficiency is a with of the correct dGTP as in a for extension of primer The are the of Taq pol I produces and at of nucleotides Kunkel T.A. PubMed Scopus Google Scholar). at a high enzyme Taq pol I is more the enzyme and primer and the frequency of A→T and In addition, at involving of the (6Suzuki M. Yoshida S. Adman E.T. Blank A. Loeb L.A. J. Biol. Chem. 2000; 275: 32728-32735Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). This study reports a forward mutation of T664P, an O-helix mutant of Taq pol I that demonstrates in replication fidelity. This mutant DNA with an increased frequency of base substitution and frameshift errors (i.e. increase in These data indicate that Taq pol I T664P is in to discriminate between correct and incorrect incoming nucleotides during DNA synthesis in Taq pol I T664P reduced replication fidelity and an altered but kinetic properties and for correct as for and primer are the same as or than wild type Taq pol I This is with the of Thr-664 for catalytic efficiency of the the amino acid corresponding to Thr-664 is the pol I of DNA polymerases. These data that the catalytic pocket of T664P a to wild type when a correct nucleotide during DNA processes to the of a polymerase to discriminate correct from incorrect nucleotides during DNA at the insertion of incoming nucleotide during and at the extension between and primer nucleotide incorporation for T664P indicate that efficiency for incorrect incorporation of dATP, and dCTP are and 10-fold higher than wild type, extension efficiency for and are and 9.5-fold higher than wild type, These data show that T664P is in at the insertion and the extension It is possible that kinetic determined a different template than the for the experiment However, this is for T664P because the data is for the forward mutation assay and the kinetic and increase is in the forward mutation assay in and base substitution the the altered replication fidelity of T664P, other mutant polymerases that have amino acid substitutions at Thr-664 were also purified and the interaction between Thr-664 and the template base plays a role in replication fidelity 1 it is that the effects of substitution of Thr-664 on the and of the amino with the amino acid reduce replication fidelity of Taq pol I in the forward mutation substitution of Thr-664 with the amino acid increases the mutation frequency a substitution of Thr-664 with in the wild type mutant frequency These data indicate that the may base selection by I, but it to a and is a amino acid because is to the a is in protein but a number of are in the regions of PubMed Scopus Google Scholar). proline for Thr-664 in the O-helix of Taq pol I, of the in the wild type to 1 this may in a steric hindrance the and may a PubMed Scopus Google Scholar, Sci. PubMed Scopus Google Scholar, J. to Scholar). data indicate that substitution of proline in the O-helix at position 664 catalytic activity for insertion of correct nucleotides by Taq pol I the hindrance in Sci. PubMed Scopus Google Scholar). One of this is that the may be with in the O-helix for incorporation of the correct of the that in the wild type enzyme. The indicate that T664P is catalytically altered during incorporation of incorrect nucleotides or when primer termini and These data that the O-helix bends and enlarges the catalytic pocket at the position of the that because of the T664P substitution In wild type Taq pol I, base pairs may the and that a in the catalytic pocket S. G. J. 1998; PubMed Scopus Google Scholar). This is to the amino and that with the substrate the O-helix a proline is near the middle of the it may a between the base and base that as an The low replication fidelity mutant of Taq pol I also (6Suzuki M. Yoshida S. Adman E.T. Blank A. Loeb L.A. J. Biol. Chem. 2000; 275: 32728-32735Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The of that study that an interaction between the O-helix and the primer might the of Taq pol I, more for and the of phosphoryl transfer involving It is also possible that amino acid substitution in the O-helix might more in the catalytic pocket to incorrect base However, the mechanisms low replication fidelity may pol I T664P and The amino acid 664 is in Pol I DNA polymerases and Pol DNA polymerases J. Nucleic Acids Res. 1993; PubMed Scopus Google Scholar). proline is at of the corresponding or in regions that to the O-helix in this group of data also that proline substitution, it during evolution of the DNA polymerases, might have excluded to ensure a sufficient of replication fidelity. in for preparation of and Blank and Adman at the of for are to at constructed a to the effects of proline substitution in the enzyme.