Comparative Kinetics of Nucleotide Analog Incorporation by Vent DNA Polymerase
2004; Elsevier BV; Volume: 279; Issue: 12 Linguagem: Inglês
10.1074/jbc.m308286200
ISSN1083-351X
AutoresAndrew F. Gardner, Catherine M. Joyce, William E. Jack,
Tópico(s)CRISPR and Genetic Engineering
ResumoComparative kinetic and structural analyses of a variety of polymerases have revealed both common and divergent elements of nucleotide discrimination. Although the parameters for dNTP incorporation by the hyperthermophilic archaeal Family B Vent DNA polymerase are similar to those previously derived for Family A and B DNA polymerases, parameters for analog incorporation reveal alternative strategies for discrimination by this enzyme. Discrimination against ribonucleotides was characterized by a decrease in the affinity of NTP binding and a lower rate of phosphoryl transfer, whereas discrimination against ddNTPs was almost exclusively due to a slower rate of phosphodiester bond formation. Unlike Family A DNA polymerases, incorporation of 9-[(2-hydroxyethoxy)methyl]X triphosphates (where X is adenine, cytosine, guanine, or thymine; acyNTPs) by Vent DNA polymerase was enhanced over ddNTPs via a 50-fold increase in phosphoryl transfer rate. Furthermore, a mutant with increased propensity for nucleotide analog incorporation (VentA488L DNA polymerase) had unaltered dNTP incorporation while displaying enhanced nucleotide analog binding affinity and rates of phosphoryl transfer. Based on kinetic data and available structural information from other DNA polymerases, we propose active site models for dNTP, ddNTP, and acyNTP selection by hyperthermophilic archaeal DNA polymerases to rationalize structural and functional differences between polymerases. Comparative kinetic and structural analyses of a variety of polymerases have revealed both common and divergent elements of nucleotide discrimination. Although the parameters for dNTP incorporation by the hyperthermophilic archaeal Family B Vent DNA polymerase are similar to those previously derived for Family A and B DNA polymerases, parameters for analog incorporation reveal alternative strategies for discrimination by this enzyme. Discrimination against ribonucleotides was characterized by a decrease in the affinity of NTP binding and a lower rate of phosphoryl transfer, whereas discrimination against ddNTPs was almost exclusively due to a slower rate of phosphodiester bond formation. Unlike Family A DNA polymerases, incorporation of 9-[(2-hydroxyethoxy)methyl]X triphosphates (where X is adenine, cytosine, guanine, or thymine; acyNTPs) by Vent DNA polymerase was enhanced over ddNTPs via a 50-fold increase in phosphoryl transfer rate. Furthermore, a mutant with increased propensity for nucleotide analog incorporation (VentA488L DNA polymerase) had unaltered dNTP incorporation while displaying enhanced nucleotide analog binding affinity and rates of phosphoryl transfer. Based on kinetic data and available structural information from other DNA polymerases, we propose active site models for dNTP, ddNTP, and acyNTP selection by hyperthermophilic archaeal DNA polymerases to rationalize structural and functional differences between polymerases. All free living organisms encode several DNA polymerases that are jointly responsible for the replication and maintenance of their genomes, thereby ensuring accurate transmission of genetic information (1Kornberg A. DNA Replication. W. H. Freeman & Co., San Francisco1980: 87-97Google Scholar, 2Joyce C.M. Steitz T.A. Annu. Rev. Biochem. 1994; 63: 777-822Crossref PubMed Scopus (570) Google Scholar, 3Steitz T.A. J. Biol. Chem. 1999; 274: 17395-17398Abstract Full Text Full Text PDF PubMed Scopus (699) Google Scholar). The majority of identified DNA polymerases can be classified into Families A, B, C, and Y according to amino acid sequence similarities to Escherichia coli polymerases I, II, III, and IV/V, respectively (4Braithwaite D.K. Ito J. Nucleic Acids Res. 1993; 21: 787-802Crossref PubMed Scopus (531) Google Scholar, 5Filée J. Forterre P. Sen-Lin T. Laurent J. J. Mol. Evol. 2002; 54: 763-773Crossref PubMed Scopus (188) Google Scholar). Additional families have been identified, including the two-subunit replicative DNA polymerases from hyperthermophilic Archaea (Family D) (6Cann I.K. Komori K. Toh H. Kanai S. Ishino Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14250-14255Crossref PubMed Scopus (118) Google Scholar) and eukaryotic DNA polymerase β and terminal transferases (Family X) (4Braithwaite D.K. Ito J. Nucleic Acids Res. 1993; 21: 787-802Crossref PubMed Scopus (531) Google Scholar). Structural and kinetic analyses of Family A (7Polesky A.H. Steitz T.A. Grindley N.D.F. Joyce C.M. J. Biol. 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Biol. 1993; 3: 31-38Crossref Scopus (201) Google Scholar). In the case of Family A DNA polymerases from bacteriophage T7, Escherichia coli (Klenow fragment, large fragment of DNA polymerase I), and Thermus aquaticus, as well as the Family B DNA polymerase from bacteriophage RB69, interpretation of the structural information is complemented by steady-state and pre-steady-state kinetic studies, allowing a detailed description of the polymerization pathway. Reaction parameters describing the discrimination against naturally occurring nucleotide analogs encountered in vivo, such as NTPs, or unnatural nucleotide analogs, such as ddNTPs and dye-labeled ddNTPs (13Brandis J.W. Edwards S.G. Johnson K.A. Biochemistry. 1996; 35: 2189-2200Crossref PubMed Scopus (43) Google Scholar, 25Yang G. Franklin M. Li J. Lin T.-C. Konigsberg W. Biochemistry. 2002; 41: 2526-2534Crossref PubMed Scopus (51) Google Scholar, 26Astatke M. Ng K. Grindley N.D.F. Joyce C.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3402-3407Crossref PubMed Scopus (182) Google Scholar, 27Astatke M. Grindley N.D.F. Joyce C.M. J. Mol. Biol. 1998; 278: 147-165Crossref PubMed Scopus (99) Google Scholar, 28Brandis J.W. Nucleic Acids Res. 1999; 27: 1912-1918Crossref PubMed Scopus (11) Google Scholar, 29Ilsley D.D. Buzby P.R. FASEB J. 1999; 13 (abstr.): A144Google Scholar, 30Yang G. Franklin M. Li J. Lin T.-C. Konigsberg W. Biochemistry. 2002; 41: 10256-10261Crossref PubMed Scopus (76) Google Scholar), have added insights into the basis for nucleotide discrimination. Hyperthermophilic archaeal DNA polymerases have not been scrutinized in such detail, hampering a complete characterization and comparison with other polymerases. Family B DNA polymerases from hyperthermophilic Archaea Thermococcus sp. 9°N (22Rodriguez A.C. Park H.-W. Mao C. Beese L.S. J. Mol. Biol. 2000; 299: 447-462Crossref PubMed Scopus (120) Google Scholar), Thermococcus gorgonarius (18Hopfner K.P. Eichinger A. Engh R.A. Laue F. 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A. 1999; 96: 3600-3605Crossref PubMed Scopus (196) Google Scholar, 29Ilsley D.D. Buzby P.R. FASEB J. 1999; 13 (abstr.): A144Google Scholar, 31Kong H. Kucera R.B. Jack W.E. J. Biol. Chem. 1993; 268: 1965-1975Abstract Full Text PDF PubMed Google Scholar, 32Gardner A.F. Jack W.E. Nucleic Acids Res. 1999; 27: 2545-2553Crossref PubMed Scopus (101) Google Scholar, 33Gardner A.F. Jack W.E. Nucleic Acids Res. 2002; 30: 605-613Crossref PubMed Scopus (65) Google Scholar, 34Evans S.J. Fogg M.J. Mamone A. Davis M. Pearl L.H. Connolly B.A. Nucleic Acids Res. 2000; 28: 1059-1066Crossref PubMed Scopus (38) Google Scholar, 35Perler F.B. Kumar S. Kong H. Adv. Protein Chem. 1996; 48: 377-435Crossref PubMed Google Scholar). Nucleotide analogs have also been important in identifying dNTP recognition determinants important in the polymerase reaction (32Gardner A.F. Jack W.E. Nucleic Acids Res. 1999; 27: 2545-2553Crossref PubMed Scopus (101) Google Scholar, 33Gardner A.F. Jack W.E. Nucleic Acids Res. 2002; 30: 605-613Crossref PubMed Scopus (65) Google Scholar, 34Evans S.J. Fogg M.J. Mamone A. Davis M. Pearl L.H. Connolly B.A. Nucleic Acids Res. 2000; 28: 1059-1066Crossref PubMed Scopus (38) Google Scholar, 35Perler F.B. Kumar S. Kong H. Adv. Protein Chem. 1996; 48: 377-435Crossref PubMed Google Scholar, 36Arezi B. Hansen C.J. Hogrefe H.H. J. Mol. Biol. 2002; 322: 719-729Crossref PubMed Scopus (11) Google Scholar) and have proven useful in a variety of molecular biology applications, such as DNA sequencing and detection of single nucleotide polymorphisms (37Sears L.E. Moran L.S. Kissinger C. Creasey T. Perry-O'Keefe H. Roskey M. Sutherland E. Slatko B.E. BioTechniques. 1992; 13: 626-633PubMed Google Scholar, 38Peterson M.G. Nucleic Acids Res. 1988; 16: 10915Crossref PubMed Scopus (12) Google Scholar, 39Bankier A.T. Methods Mol. Biol. 1993; 23: 83-90PubMed Google Scholar, 40Prober J.M. Trainor G.L. Dam R.J. Hobbs F.W. Robertson C.W. Zagursky R.J. Cocuzza A.J. Jensen M.A. Baumeister K. Science. 1987; 238: 336-341Crossref PubMed Scopus (691) Google Scholar, 41Chen X. Levine L. Kwok P.Y. Genome Res. 1999; 9: 492-498PubMed Google Scholar). One group of analogs, 9-[(2-hydroxyethoxy)methyl]X triphosphates (where X is adenine, cytosine, guanine, or thymine; acyNTPs), 1The abbreviations used are: acyNTP, 9-[(2-hydroxyethoxy)methyl]X triphosphate, where X is adenine, cytosine, guanine, or thymine; acyCTP, 9-[(2-hydroxyethoxy)methyl)]cytosine triphosphate; dCTPαS, 2′-deoxycytidine 5′-O-(1-thiotriphosphate); ddCTPαS, 2′,3′-dideoxycytidine 5′-O-(1-thiotriphosphate); ROX-, 6-carboxy-X-rhodamine; FAM, 6-carboxyfluorescein. is particularly intriguing due to the wide spectrum of incorporation efficiency noted in different DNA polymerases, even within the same family of polymerase. For example, within Family B, the herpes simplex virus type 2 and human cytomegalovirus DNA polymerases incorporate acyNTPs more efficiently than ddNTPs, whereas human polymerase α more readily inserts ddNTPs over acyNTPs (42Reid R. Mar E.C. Huang E.S. Topal M.D. J. Biol. Chem. 1988; 263: 3898-3904Abstract Full Text PDF PubMed Google Scholar). Such differences have been exploited in drug therapies where infective agents encode polymerases that more readily insert acyNTP than does the host DNA polymerase (43Freeman S. Gardiner J.M. Mol. Biotechnol. 1996; 5: 125-137Crossref PubMed Scopus (58) Google Scholar). Hyperthermophilic archaeal DNA polymerases (Vent®, Deep Vent™, 9°N™, and Pfu) all incorporate acyNTPs with greater efficiency than ddNTPs (33Gardner A.F. Jack W.E. Nucleic Acids Res. 2002; 30: 605-613Crossref PubMed Scopus (65) Google Scholar), in contrast with the behavior of Taq and Klenow fragment DNA polymerases, which prefer ddNTPs (33Gardner A.F. Jack W.E. Nucleic Acids Res. 2002; 30: 605-613Crossref PubMed Scopus (65) Google Scholar, 44Trainor G.L. U. S. Patent 5,558,991. February 7, 1996; Google Scholar). In the course of probing the determinants of nucleotide sugar discrimination in the Family B DNA polymerase from the hyperthermophilic Archaea Thermococcus litoralis (Vent DNA polymerase), we identified a mutant (VentA488L DNA polymerase) that reduces discrimination against several altered nucleotides (32Gardner A.F. Jack W.E. Nucleic Acids Res. 1999; 27: 2545-2553Crossref PubMed Scopus (101) Google Scholar, 33Gardner A.F. Jack W.E. Nucleic Acids Res. 2002; 30: 605-613Crossref PubMed Scopus (65) Google Scholar). Subsequent crystal structures of closely related DNA polymerases strongly suggested that this residue makes neither direct nor indirect contacts with the reaction substrates, raising questions about the structural basis for the observed variation (Fig. 1B). The universality of the A488L phenotype was later confirmed by homologous mutations in other hyperthermophilic DNA polymerases (Pfu A486Y DNA polymerase (34Evans S.J. Fogg M.J. Mamone A. Davis M. Pearl L.H. Connolly B.A. Nucleic Acids Res. 2000; 28: 1059-1066Crossref PubMed Scopus (38) Google Scholar), 9°N A485L DNA polymerase (33Gardner A.F. Jack W.E. Nucleic Acids Res. 2002; 30: 605-613Crossref PubMed Scopus (65) Google Scholar), and Tsp JDF-3 A485T DNA polymerase (36Arezi B. Hansen C.J. Hogrefe H.H. J. Mol. Biol. 2002; 322: 719-729Crossref PubMed Scopus (11) Google Scholar)), further emphasizing a conserved role for this residue. Although instructive, these steady-state observations failed to address the underlying kinetic mechanisms responsible for nucleotide and nucleotide analog incorporation in hyperthermophilic DNA polymerases. Therefore, we initiated pre-steady-state kinetic studies to compare the modes of nucleotide discrimination in Vent and other DNA polymerases. Nucleotides, Nucleotide Analogs, DNA Substrate, and Enzymes—All DNA polymerases used in this study are 3′ → 5′ exonuclease-deficient as a result of mutation of catalytic aspartic and glutamic acids to alanine in the exonuclease active site (31Kong H. Kucera R.B. Jack W.E. J. Biol. Chem. 1993; 268: 1965-1975Abstract Full Text PDF PubMed Google Scholar, 32Gardner A.F. Jack W.E. Nucleic Acids Res. 1999; 27: 2545-2553Crossref PubMed Scopus (101) Google Scholar, 45Derbyshire 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). These mutations prevent exonuclease removal of newly incorporated nucleotides or terminators. Vent and VentA488L DNA polymerases were purified as described previously (31Kong H. Kucera R.B. Jack W.E. J. Biol. Chem. 1993; 268: 1965-1975Abstract Full Text PDF PubMed Google Scholar), and the concentration was determined spectroscopically at 280 nm using an extinction coefficient of 115,960 liter mol-1 cm-1. The concentration of E. coli DNA polymerase I (Klenow fragment exo-; New England Biolabs Inc., Beverly, MA) was calculated using a specific activity of 20,000 units/mg. dNTPs, ddCTP, and 9-[(2-hydroxyethoxy)methyl)]cytosine triphosphate (acyCTP) were from New England Biolabs Inc. 2′-Deoxycytidine 5′-O-(1-thiotriphosphate) (dCTPαS) and CTP were from Amersham Biosciences. 2′,3′-Dideoxycytidine 5′-O-(1-thiotriphosphate) (ddCTPαS) was from TriLink BioTechnologies (San Diego, CA). 6-Carboxy-X-rhodamine (ROX)-derivatized nucleotide analogs ROX-ddCTP and ROX-acyCTP were kindly provided by Phil Buzby (PerkinElmer Life Sciences) (Fig. 2). Oligonucleotides used to measure 2′-deoxycytosine 5′-triphosphate (dCTP) and cytosine analog incorporation were synthesized and purified by the Oligonucleotide Synthesis Division at New England Biolabs with a 6-carboxyfluorescein (FAM) label on the primer strand for detection: 5′-FAM-CCCTCGCAGCCGTCCAACCAACTCA-3′ (25-mer) and 3′-GGGAGCGTCGGCAGGTTGGTTGAGTGCCTCTTGTTT-5′ (36-mer). FAM-duplex DNA was formed by mixing equimolar amounts of the dye-labeled 25-mer primer with the 36-mer template in annealing buffer (5 mm Tris-HCl (pH 8.0 at 20 °C), 5 mm NaCl, and 0.2 mm EDTA) and heating the solutions to 95 °C for 5 min, followed by incubation for 10 min at 60 °C and then cooling for 15 min at room temperature. Burst Kinetics and Active Site Titration—To measure the fraction of active Vent DNA polymerase and to determine the position of the rate-limiting step within the polymerase reaction pathway, we investigated whether the reaction followed burst kinetics. Rapid quench reactions were carried out as described below with 50 nm FAM-duplex DNA; 10 or 20 nm Vent or VentA488L DNA polymerase; and 0.20 mm dCTP, ddCTP, ddCTPαS, CTP, or acyCTP (final concentrations after mixing) in 1× ThermoPol buffer (10 mm KCl, 20 mm Tris-HCl (pH 8.8 at 25 °C), 10 mm (NH4)2SO4, 2 mm MgSO4, and 0.1% Triton X-100). The steady-state rate (k2), the burst amplitude (A, which is equal to the active site concentration), and the initial rate of product formation (r, the burst rate) were extrapolated from the burst equation: [product] = A(1 - exp-rt) + k2t (45Derbyshire 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). The steady-state turnover number (kSS) was calculated by dividing k2 by A. Measurement of DNA Polymerase Pre-steady-state Kinetic Parameters—Single turnover nucleotide incorporation reactions were initiated by mixing Vent or VentA488L DNA polymerase (0.10 μm) and FAM-duplex DNA (0.050 μm) in 1× ThermoPol buffer together with an equal volume of nucleotides or nucleotide analogs in 1× ThermoPol buffer. The reactions were allowed to proceed for the indicated times and then quenched by addition of EDTA to a final concentration of 0.35-0.40 m. Reactions in the range of 3 ms to 10 s were sampled using an RQF-3 rapid quenched-flow instrument (Kintek Corp., Austin, TX). Reactions with an initial time point >10 s were mixed and quenched manually. All Vent DNA polymerase reactions were analyzed at 60 °C. Although this temperature is lower than the optimal reaction temperature of 72 °C (31Kong H. Kucera R.B. Jack W.E. J. Biol. Chem. 1993; 268: 1965-1975Abstract Full Text PDF PubMed Google Scholar), it is the highest temperature at which the rapid quench instrument can be operated reliably. Single turnover acyCTP incorporation by Klenow fragment DNA polymerase was initiated by mixing 1.0 μm Klenow fragment DNA polymerase (exo-) and 0.10 μm FAM-duplex DNA substrate in 1× Klenow buffer (50 mm Tris-HCl (pH 7.5) and 2 mm MgCl2) with an equal volume of acyCTP in 1× Klenow buffer. Reactions were then incubated at 25 °C for various times and quenched manually with EDTA (0.1 m final concentration). Conversion of the fluorescently labeled DNA primer-template to product was monitored by denaturing PAGE and automated fluorescence detection methods. Product DNA was denatured by mixing a 7.5 μl aliquot of quenched sample with 45 μl of formamide and 1.5 mm EDTA and heating at 90 °C for 3 min. Fluorescent 5′-FAM-labeled 25-mer oligonucleotide substrate and 5′-FAM-labeled 26-mer oligonucleotide product bands were fractionated by electrophoresis on an 8.8 m urea and 16% polyacrylamide denaturing gel using an ABI377 automated sequencer (Applied Biosystems, Foster City, CA) and quantified using GeneScan Version 2.1 software (Applied Biosystems). The first-order rate constant for polymerase-catalyzed addition at each nucleotide concentration was calculated from a plot of ln[substrate] versus time. Rate constants (kobs) were subsequently plotted as a function of nucleotide or analog concentration and fitted to the hyperbolic equation: kobs = (kpol[nucleotide])/(KD + [nucleotide]), yielding kpol, the maximum rate of nucleotide addition, and KD, the dissociation constant for nucleotide binding (46Johnson K.A. Methods Enzymol. 1995; 249: 38-61Crossref PubMed Scopus (194) Google Scholar). The activation energy difference between dNTP and nucleotide analog incorporation was calculated by Equation 1 (47Fersht A.R. Shi J.P. Knill-Jones J. Lowe D.M. Wilkinson A.J. Blow D.M. Brick P. Carter P. Waye M.M. Winter G. Nature. 1985; 314: 235-238Crossref PubMed Scopus (993) Google Scholar). ΔΔG‡=-RTln((kpol/KD)dNTP/(kpol/KD)nucleotideanalog)(Eq. 1) Single turnover kinetics require saturating enzyme concentrations. We established that 0.10 μm Vent DNA polymerase was sufficient under the reaction conditions described by demonstrating that the rates of ddCTP incorporation were the same using Vent DNA polymerase concentrations of 0.10, 0.20, and 0.40 μm (data not shown). Measurement of Pyrophosphorolysis Catalyzed by Vent DNA Polymerase—To measure the rate of DNA degradation by pyrophosphorolysis, Vent or VentA488L DNA polymerase (0.10 μm) was equilibrated with the DNA substrate (0.050 μm) in 1× ThermoPol buffer and then mixed with PPi in 1× ThermoPol buffer at 60 °C using rapid quench techniques as described above. The extent of pyrophosphorolysis at each time point was calculated by multiplying the mole fraction of each DNA species by the number of phosphodiester bonds hydrolyzed to generate that species. KD(PPi) and kpyro were derived using fitting protocols analogous to those described above for nucleotide addition. Analysis of dNTP Incorporation by Vent DNA Polymerase—Previous studies with Family A DNA polymerases have shown that the steady-state rate-limiting step for addition of a single correctly paired dNTP follows phosphodiester bond formation (8Patel S.S. Wong I. Johnson K.A. Biochemistry. 1991; 30: 511-525Crossref PubMed Scopus (473) Google Scholar, 13Brandis J.W. Edwards S.G. Johnson K.A. Biochemistry. 1996; 35: 2189-2200Crossref PubMed Scopus (43) Google Scholar, 46Johnson K.A. Methods Enzymol. 1995; 249: 38-61Crossref PubMed Scopus (194) Google Scholar, 48Benkovic S.J. Cameron C.E. Methods Enzymol. 1995; 262: 257-269Crossref PubMed Scopus (48) Google Scholar). Consequently, the first round of polymerization occurs more rapidly than subsequent rounds, resulting in a rapid initial burst of product. Incorporation of dCTP by Vent DNA polymerase displayed a burst pattern similar to those seen with RB69 and AmpliTaq-CS DNA polymerases, with a rapid burst (kburst = 60 s-1) followed by slow steady-state turnover (kSS = 0.90 s-1) (Fig. 3A and Table I). As indicated above, the burst is diagnostic for a rate-limiting step following bond formation; moreover, its amplitude is equal to the concentration of active enzyme, indicating that >90% of the Vent DNA polymerase preparation was active. Under similar conditions, Vent DNA polymerase failed to show a significant burst with ddCTP (Fig. 3B) or CTP (data not shown) incorporation. These data suggest that the rate-limiting step during nucleotide analog incorporation has changed compared with dNTP. Upon substitution of ddCTP with ddCTPαS, both Vent and VentA488L DNA polymerases showed a 10- and 6-fold thio elemental effect (kburst(ddCTP)/kburst(ddCTPαS)), respectively (Table I), consistent with an altered rate-limiting step.Table IPre-steady-state burst kineticsEnzymekburst(dCTP)kSS(dCTP)kburst(ddCTP)kburst(ddCTPαS)s−1s−1s−1s−1Vent60 ± 400.90 ± 0.090.47 ± 0.050.044 ± 0.024VentA488L45 ± 90.10 ± 0.010.23 ± 0.020.039 ± 0.013RB69aRef. 25.230 ± 402.7 ± 0.2NDNDAmpliTaq-CSbRef. 13.50 ± 72.5 ± 0.2NDNDa Ref. 25Yang G. Franklin M. Li J. Lin T.-C. Konigsberg W. Biochemistry. 2002; 41: 2526-2534Crossref PubMed Scopus (51) Google Scholar.b Ref. 13Brandis J.W. Edwards S.G. Johnson K.A. Biochemistry. 1996; 35: 2189-2200Crossref PubMed Scopus (43) Google Scholar. Open table in a new tab Determinations of KD and kpol for dCTP addition by Vent DNA polymerase gave kinetic constants similar to those determined for other DNA polymerases (Fig. 4A and Tables II and III). The relatively high KD for nucleotides (KD = 70 μm) is similar to the Km for nucleotides determined in multiple turnover steady-state measurements (Km = 40 μm) (31Kong H. Kucera R.B. Jack W.E. J. Biol. Chem. 1993; 268: 1965-1975Abstract Full Text PDF PubMed Google Scholar). Kinetic constants show little dependence on nucleotide identity, as similar Vent DNA polymerase binding (KD = 58 μm) and rate (kpol = 64 s-1) constants were observed for dATP incorporation. Substitution of dCTP with dCTPαS had little effect on binding (KD) or phosphodiester bond formation (kpol); thus, the polymerase displays a minimum thio elemental effect (kpol(dCTP)/kpol(dCTPαS) = 0.80) (Table II).Table IIPre-steady-state kinetic constants for nucleotide and nucleotide analog incorporation by Vent and VentA488L DNA polymerasesNucleotideVent DNA polymeraseVentA488L DNA polymeraseKDkpolkpol/KDSelectivityaSelectivity between incorporation of dCTP and other NTPs is the ratio of the efficiency of dCTP incorporation (kpol/KD) to the efficiency of CTP, ddCTP, or acyCTP incorporation.KDkpolkpol/KDSelectivityaSelectivity between incorporation of dCTP and other NTPs is the ratio of the efficiency of dCTP incorporation (kpol/KD) to the efficiency of CTP, ddCTP, or acyCTP incorporation.μms−1m−1 s−1μms−1m−1 s−1dCTP70 ± 766 ± 19.5 × 10577 ± 956 ± 37.3 × 105dCTPαS120 ± 4082 ± 136.6 × 1051.468 ± 5328.0 ± 0.34.1 × 1051.8CTP1100 ± 1000.160 ± 0.0051.5 × 1026000360 ± 300.70 ± 0.201.6 × 103450ddCTP46 ± 70.16 ± 0.013.5 × 10327018 ± 60.30 ± 0.021.8 × 10440acyCTP81 ± 257.6 ± 1.19.7 × 1041024.0 ± 0.413 ± 25.4 × 1051.4ROX-ddCTP10 ± 20.029 ± 0.0032.9 × 1033256.0bThe kinetic parameters for VentA488L DNA polymerase are from single determinations.0.2bThe kinetic parameters for VentA488L DNA polymerase are from single determinations.2.5 × 10430ROX-acyCTP8.0 ± 0.22.00 ± 0.012.5 × 10546bThe kinetic parameters for VentA488L DNA polymerase are from single determinations.1bThe kinetic parameters for VentA488L DNA polymerase are from single determinations.1.6 × 1054.5a Selectivity between incorporation of dCTP and other NTPs is the ratio of the efficiency of dCTP incorporation (kpol/KD) to the efficiency of CTP, ddCTP, or acyCTP incorporation.b The kinetic parameters for VentA488L DNA polymerase are from single determinations. Open table in a new tab Table IIIPre-steady-state kinetic constants for nucleotide analog incorporation by DNA polymerasesEnzymedCTPCTPddCTPacyCTPKDkpolKDkpolSelectivityaSelectivity between incorporation of dCTP and other NTPs is the ratio of the efficiency of dCTP incorporation (kpol/KD) to the efficiency of CTP, ddCTP, or acyCTP incorporation.KDkpolSelectivityKDkpolSelectivityaSelectivity between incorporation of dCTP and other NTPs is the ratio of the efficiency of dCTP incorporation (kpol/KD) to the efficiency of CTP, ddCTP, or acyCTP incorporation.μms−1μms−1μms−1μms−1Vent70 ± 766 ± 11100 ± 1000.160 ± 0.005600046 ± 70.16 ± 0.0127081 ± 257.6 ± 1.110VentA488L77 ± 956 ± 3360 ± 300.70 ± 0.2045018 ± 60.30 ± 0.024024.0 ± 0.413 ± 21.4RB6969 ± 16bRef. 30.200 ± 13bRef. 30.16,000 ± 400bRef. 30.0.74 ± 0.2bRef. 30.64,000bRef. 30.4300 ± 800bRef. 30.0.17 ± 0.02bRef. 30.73,000bRef. 30.NDNDNDKlenow9.6 ± 2.3cRef. 26.75 ± 13cRef. 26.21 ± 7cRef. 26.0.047 ± 0.025cRef. 26.3400cRef. 26.8.4 ± 4dRef. 27.0.015 ± 0.004dRef. 27.4200dRef. 27.200 ± 300.048 ± 0.00432,000KlenTaq35 ± 2eRef. 13.21 ± 4eRef. 13.NDNDND58 ± 10eRef. 13.0.03 ± 0.003eRef. 13.1200eRef. 13.NDNDNDa Selectivity between incorporation of dCTP and other NTPs is the ratio of the efficiency of dCTP incorporation (kpol/KD) to the efficiency of CTP, ddCTP, or acyCTP incorporation.b Ref. 30Yang G. Franklin M. Li J. Lin T.-C. Konigsberg W. Biochemistry. 2002; 41: 10256-10261Crossref PubMed Scopus (76) Google Scholar.c Ref. 26Astatke M. Ng K. Grindley N.D.F. Joyce C.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3402-3407Crossref PubMed Scopus (182) Google Scholar.d Ref. 27Astatke M. Grindley N.D.F. Joyce
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