The Gly-952 Residue of Saccharomyces cerevisiae DNA Polymerase α Is Important in Discriminating Correct Deoxyribonucleotides from Incorrect Ones
2003; Elsevier BV; Volume: 278; Issue: 21 Linguagem: Inglês
10.1074/jbc.m208604200
ISSN1083-351X
AutoresSiripan Limsirichaikul, Masanori Ogawa, Atsuko Niimi, Shigenori Iwai, Takashi Murate, Shonen Yoshida, Motoshi Suzuki,
Tópico(s)Genomics and Chromatin Dynamics
ResumoGly-952 is a conserved residue in Saccharomyces cerevisiae DNA polymerase α (pol α) that is strictly required for catalytic activity and for genetic complementation of a pol α-deficient yeast strain. This study analyzes the role of Gly-952 by characterizing the biochemical properties of Gly-952 mutants. Analysis of the nucleotide incorporation specificity of pol α G952A showed that this mutant incorporates nucleotides with extraordinarily low fidelity. In a steady-state kinetic assay to measure nucleotide misincorporation, pol α G952A incorporated incorrect nucleotides more efficiently than correct nucleotides opposite template C, G, and T. The fidelity of the G952A mutant polymerase was highest at template A, where the ratio of incorporation of dCMP to dTMP was as high as 0.37. Correct nucleotide insertion was 500- to 3500-fold lower for G952A than for wild type pol α, with up to 22-fold increase in pyrimidine misincorporation. The Km for G952A pol α bound to mismatched termini T:T, T:C, C:A, and A:C was 71- to 460-fold lower than to a matched terminus. Furthermore, pol α G952A preferentially incorporated pyrimidine instead of dAMP opposite an abasic site, cis-syn cyclobutane di-thymine, or (6–4) di-thymine photoproduct. These data demonstrate that Gly-952 is a critical residue for catalytic efficiency and error prevention in S. cerevisiae pol α. Gly-952 is a conserved residue in Saccharomyces cerevisiae DNA polymerase α (pol α) that is strictly required for catalytic activity and for genetic complementation of a pol α-deficient yeast strain. This study analyzes the role of Gly-952 by characterizing the biochemical properties of Gly-952 mutants. Analysis of the nucleotide incorporation specificity of pol α G952A showed that this mutant incorporates nucleotides with extraordinarily low fidelity. In a steady-state kinetic assay to measure nucleotide misincorporation, pol α G952A incorporated incorrect nucleotides more efficiently than correct nucleotides opposite template C, G, and T. The fidelity of the G952A mutant polymerase was highest at template A, where the ratio of incorporation of dCMP to dTMP was as high as 0.37. Correct nucleotide insertion was 500- to 3500-fold lower for G952A than for wild type pol α, with up to 22-fold increase in pyrimidine misincorporation. The Km for G952A pol α bound to mismatched termini T:T, T:C, C:A, and A:C was 71- to 460-fold lower than to a matched terminus. Furthermore, pol α G952A preferentially incorporated pyrimidine instead of dAMP opposite an abasic site, cis-syn cyclobutane di-thymine, or (6–4) di-thymine photoproduct. These data demonstrate that Gly-952 is a critical residue for catalytic efficiency and error prevention in S. cerevisiae pol α. DNA polymerases belong to at least five classes, whose members share a high degree of amino acid sequence similarity (1Burgers P.M. Koonin E.V. Bruford E. Blanco L. Burtis K.C. Christman M.F. Copeland W.C. Friedberg E.C. Hanaoka F. Hinkle D.C. Lawrence C.W. Nakanishi M. Ohmori H. Prakash L. Prakash S. Reynaud C.A. Sugino A. Todo T. Wang Z. Weill J.C. Woodgate R. J. Biol. Chem. 2001; 276: 43487-43490Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 2Friedberg E.C. Feaver W.J. Gerlach V.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5681-5683Crossref PubMed Scopus (226) Google Scholar, 3Steitz T.A. J. Biol. Chem. 1999; 274: 17395-17398Abstract Full Text Full Text PDF PubMed Scopus (712) Google Scholar). Polymerase classes include class A (e.g. Escherichia coli pol 1The abbreviations used are: pol, DNA polymerase; BSA, bovine serum albumin; DTT, dithiothreitol; CPD, cis-syn cyclobutane di-thymine. I, Taq pol I, pol γ), class B (e.g. pol α, RB69 pol), class C (e.g. pol III), class X (e.g. pol β), and class Y (e.g. pol η). Despite the fact that the consensus sequence of each polymerase class is significantly different from the consensus sequences of other polymerase classes, the tertiary structures of all polymerases are highly similar. The crystal structures of many polymerases have a common architecture, which has been described as a right hand composed of three domains corresponding to the palm, fingers, and thumb (3Steitz T.A. J. Biol. Chem. 1999; 274: 17395-17398Abstract Full Text Full Text PDF PubMed Scopus (712) Google Scholar). These subdomains form a DNA binding track and the catalytic pocket. When nucleotides are incorporated into DNA during catalysis, the fingers subdomain rotates from an open to a closed position that faces the incoming dNTP (4Doublie S. Tabor S. Long A.M. Richardson C.C. Ellenberger T. 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Class A DNA polymerases have a conserved sequence called motif B with the amino acid sequence, R—K—F—YG, which lies in the fingers subdomain and faces toward the catalytic site. In the open complex, Tyr is positioned exactly where template base is expected for maintaining the stacking interactions with the incoming dNTP, whereas in the closed complex, the substrate base is held in position by the Phe residue. Basic amino acids, Arg and Lys, interact with the triphosphate moiety of the incoming nucleotide (4Doublie S. Tabor S. Long A.M. Richardson C.C. Ellenberger T. Nature. 1998; 391: 251-258Crossref PubMed Scopus (1117) Google Scholar, 5Kiefer J.R. Mao C. Braman J.C. Beese L.S. Nature. 1998; 391: 304-307Crossref PubMed Scopus (489) Google Scholar, 6Li Y. Korolev S. Waksman G. EMBO J. 1998; 17: 7514-7525Crossref PubMed Scopus (670) Google Scholar, 10Suzuki M. Baskin D. Hood L. Loeb L.A. Proc. Natl. Acad. Sci. U. S. 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Phe-667 in Taq pol I (and the equivalent residue in Klenow and T7 pol) is also critical for polymerase activity and for discrimination of 2′,3′-dideoxynucleotides (11Astatke M. Grindley N.D.F. Joyce C.M. J. Biol. Chem. 1995; 270: 1945-1954Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 13Astatke M. Grindley N.D. Joyce C.M. J. Mol. Biol. 1998; 278: 147-165Crossref PubMed Scopus (99) Google Scholar, 14Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6339-6343Crossref PubMed Scopus (310) Google Scholar). Tyr-766 in Klenow fragment (Tyr-671 in Taq pol I) plays a role in fidelity of DNA synthesis (15Bell 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, 16Carroll S.S. Cowart M. Benkovic S.J. Biochemistry. 1991; 30: 804-813Crossref PubMed Scopus (116) Google Scholar). Recently, Ponamarev et al. (17Ponamarev M.V. Longley M.J. Nguyen D. Kunkel T.A. Copeland W.C. J. Biol. Chem. 2002; 277: 15225-15228Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) reported that a human pol γ mutant in which a Tyr equivalent to Tyr-671 is changed to Cys has a higher Km and lower fidelity than wild type (17Ponamarev M.V. Longley M.J. Nguyen D. Kunkel T.A. Copeland W.C. J. Biol. Chem. 2002; 277: 15225-15228Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Interestingly, these aromatic resides seem to interact differently with template (Tyr-671) or substrate (Phe-667) in the open and closed structures, which suggests that they might function as a molecular chaperon (4Doublie S. Tabor S. Long A.M. Richardson C.C. Ellenberger T. Nature. 1998; 391: 251-258Crossref PubMed Scopus (1117) Google Scholar, 5Kiefer J.R. Mao C. Braman J.C. Beese L.S. Nature. 1998; 391: 304-307Crossref PubMed Scopus (489) Google Scholar, 6Li Y. Korolev S. Waksman G. EMBO J. 1998; 17: 7514-7525Crossref PubMed Scopus (670) Google Scholar, 18Suzuki M. 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Nucleic Acids Res. 2001; 29: 4206-4214Crossref PubMed Google Scholar). Thus motif B is important for catalytic activity and fidelity of DNA synthesis in class A DNA polymerases. In class B DNA polymerases, mutants at the Lys residues corresponding to Taq pol I Lys-663 demonstrate large increases in Km for the incoming nucleotide (27Dong Q. Wang T.S. J. Biol. Chem. 1995; 270: 21563-21570Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 28Saturno J. Lazaro J.M. Esteban F.J. Blanco L. Salas M. J. Mol. Biol. 1997; 269: 313-325Crossref PubMed Scopus (25) Google Scholar, 29Yang G. Franklin M. Li J. Lin T.C. Konigsberg W. Biochemistry. 2002; 41: 2526-2534Crossref PubMed Scopus (51) Google Scholar). However, compared with class A DNA polymerases, less is known about the function of other motif B residues in class B DNA polymerases. Class B DNA polymerases share a motif in the P helix that is similar to motif B in the O helix of class A DNA polymerases and has the sequence Q—K—N–YG. This motif in the P helix and residues in the N helix may together perform a comparable function to motif B (30Franklin M.C. Wang J. Steitz T.A. Cell. 2001; 105: 657-667Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar, 31Yang G. Lin T. Karam J. Konigsberg W.H. Biochemistry. 1999; 38: 8094-8101Crossref PubMed Scopus (45) Google Scholar). In the P helix of class B DNA polymerases, the distance between the Lys and Tyr is one residue shorter than in the O helix of class A DNA polymerases (32Ogawa M. Limsirichaikul S. Niimi A. Iwai S. Yoshida S. Suzuki M. J. Biol. Chem. 2003; 278: 19071-19078Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). As a result, the conserved residues in the putative α helices do not have the same spatial relationships among class A and B polymerases and may be displaced by an approximately [1/4] helical turn. In the accompanying article (32Ogawa M. Limsirichaikul S. Niimi A. Iwai S. Yoshida S. Suzuki M. J. Biol. Chem. 2003; 278: 19071-19078Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar), data are presented suggesting that conserved Gly-952 in Saccharomyces cerevisiae pol α may play a similar role to motif B residue Tyr-951 in class A DNA polymerases. This idea is consistent with the hypothesis that the active sites of class A and B DNA polymerases are structurally distinct (32Ogawa M. Limsirichaikul S. Niimi A. Iwai S. Yoshida S. Suzuki M. J. Biol. Chem. 2003; 278: 19071-19078Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). This manuscript presents steady-state kinetic analysis of pol α G952A and G952Y and shows that Gly-952 is a critical residue for correct nucleotide incorporation by S. cerevisiae pol α. Parameters that influence polymerase fidelity include template-substrate interaction (33Kunkel T.A. Bebenek K. Annu. Rev. Biochem. 2000; 69: 497-529Crossref PubMed Scopus (812) Google Scholar), stability of the catalytic complex (18Suzuki 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 (66) Google Scholar, 26Yoshida K. Tosaka A. Kamiya H. Murate T. Kasai H. Nimura Y. Ogawa M. Yoshida S. Suzuki M. Nucleic Acids Res. 2001; 29: 4206-4214Crossref PubMed Google Scholar, 34Shah A.M. Conn D.A. Li S.X. Capaldi A. Jager J. Sweasy J.B. Biochemistry. 2001; 40: 11372-11381Crossref PubMed Scopus (25) Google Scholar), and efficiency of correct nucleotide incorporation (35Shah A.M. Li S.X. Anderson K.S. Sweasy J.B. J. Biol. Chem. 2001; 276: 10824-10831Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 36Beard W.A. Shock D.D. Van de Berg B.J. Wilson S.H. J. Biol. Chem. 2002; 277: 47393-47398Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). This study demonstrates that correct nucleotide incorporation efficiency is altered in pol α G952A producing a dramatic loss of DNA polymerase fidelity. Thus, this mutant demonstrates that efficiency of correct nucleotide incorporation is directly related to polymerase fidelity. Site-directed Mutagenesis and Enzyme Preparation—Expression, purification, and specific activity determination of recombinant pol α are described (32Ogawa M. Limsirichaikul S. Niimi A. Iwai S. Yoshida S. Suzuki M. J. Biol. Chem. 2003; 278: 19071-19078Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Primer Extension Assay—Primer extension assay was performed as described previously (24Suzuki 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) with slight modifications. The 14-mer DNA primer, 5′-CGC GCC GAA TTC CC-3′ was 5′-32P-labeled, annealed to the 46-mer DNA template strand, 5′-GCG CGG AAG CTT GGC TGC AGA ATA TTG CTA GCG GGA ATT CGG CGC G-3′. The reaction was carried out in a volume of 25 μl containing 10 nm template-primer, 100 mm Tris-HCl, pH 8.0, 5 mm MgCl2, 50 mm KCl, 100 ng/ml BSA, 2 mm DTT, 1250 μm dNTP, and 10 nm wild type pol α, 30 nm G952Y, or 70 nm G952A. Reactions were incubated at 37 °C for 10 min. The amount of enzyme in the reaction was adjusted such that 80–90% of the primer is utilized in the presence of correct nucleotide dGMP. Processivity Assay—An oligo(dT)16 (Amersham Biosciences) was 32P-labeled at its 5′ terminus and annealed to poly(dA) (Amersham Biosciences) at a weight ratio of 1:10. DNA polymerase was incubated at 37 °C for 10 min in a 25-μl reaction containing 100 mm Tris-HCl, pH 8.0, 5 mm MgCl2,50mm KCl, 100 ng/μl BSA,2mm DTT, 1.25 mm dTTP, 40 ng/μl template annealed to 4 ng/μl primer; enzyme concentration was varied to optimize the assay. Reactions were terminated by addition of an equal volume of termination buffer (98% formamide, 10 mm EDTA, pH 8.0, 0.05% bromphenol blue, 0.05% xylene cyanol) and analyzed by 14% denaturing acrylamide gel electrophoresis. The products were quantified using a laser image analyzer (BAS2000 system; Fuji Film, Tokyo, Japan). Single Nucleotide Incorporation Kinetics—Steady-state kinetic constants were determined for incorporation of correct and incorrect nucleotides (Ultrapure dNTP; Amersham Biosciences). 32P-Labeled 14-mer (5′-CGC GCC GAA TTC CC-3′), 15-mer (5′-CGC GCC GAA TTC CCG-3′), 16-mer (5′-CGC GCC GAA TTC CCG C-3′), or 17-mer (5′-CGC GCC GAA TTC CCG CT-3′) primer was annealed to 46-mer of oligonucleotide, 5′-GCG CGG AAG CTT GGC TGC AGA ATA TTG CTAGCG GGA ATT CGG CGC G-3′, where underlined nucleotides were the target template sites. Reaction conditions were used such that 20% of template-primer was utilized (37Goodman M.F. Creighton S. Bloom L.B. Petruska J. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 83-126Crossref PubMed Scopus (407) Google Scholar). The reaction mixture contained 100 mm Tris-HCl, pH 8.0, 5 mm MgCl2, 50 mm KCl, 100 ng/μl BSA, 2 mm DTT, 10 nm template-primer, various concentrations of dNTP, and the optimum concentration of DNA polymerase. Reaction was incubated at 37 °C for 5 (wild type) or 10 min (mutants) in a final volume of 20 μl or with appropriate changes to obtain the proper reaction efficiency and substrate utilization. Reactions were terminated and analyzed as described above, using data from at least three experiments. Kinetic parameters (Km and kcat) for incorporation of each dNMP were determined by Hanes-Woolf plots. Single Nucleotide Extension Kinetics—Kinetics were examined from mismatched termini as follows. 32P-Labeled 15-mer (5′-CGC GCC GAA TTC CCN-3′), 16-mer (5′-CGC GCC GAA TTC CCG N-3′), 17-mer (5′-CGC GCC GAA TTC CCG CN-3′), or 18-mer (5′-CGC GCC GAA TTC CCG CTN-3′) primers were annealed to the 46-mer oligonucleotide, 5′-GCG CGG AAG CTT GGC TGC AGA ATA TTG CTAGCG GGA ATT CGG CGC G-3′, where N represents either one of the four nucleotides, and the underlined nucleotides were the target template sites. The reaction mixture (20 μl) contained 100 mm Tris-HCl, pH 8.0, 5 mm MgCl2, 50 mm KCl, 100 ng/μl BSA, 2 mm DTT, 10 nm template-primer, various concentrations of dNTP, and an optimum concentration of each enzyme. The mixture was incubated at 37 °C for 20 min (wild type) or 60 min (G952A). Reaction products were analyzed as described above, using data from at least three experiments. Under the conditions employed, reaction product increased linearly for up to an hour for both wild type and G952A pol α. Enzyme Dissociation Constants—Apparent Km and kcat were determined as described above except that the DNA concentration was varied from 10 to 200 nm. KD (DNA) values were determined with DNA concentration of 40 and 200 nm using the following equation, KD = [Dlow](kcat/Km)high/(kcat/Km)low (38Creighton S. Huang M.M. Cai H. Arnheim N. Goodman M.F. J. Biol. Chem. 1992; 267: 2633-2639Abstract Full Text PDF PubMed Google Scholar), in which kcat/Km, is an average of data from three independent experiments. Relative dissociation constant, KD(rel), was determined using the equilibrium binding method (32Ogawa M. Limsirichaikul S. Niimi A. Iwai S. Yoshida S. Suzuki M. J. Biol. Chem. 2003; 278: 19071-19078Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 38Creighton S. Huang M.M. Cai H. Arnheim N. Goodman M.F. J. Biol. Chem. 1992; 267: 2633-2639Abstract Full Text PDF PubMed Google Scholar) as follows: KD(rel) = KD(challenge dna)/KD(template-primer dna) = % extension(challenge dna)/[2 × % extension(template-primer dna) – % extension(challenge dna)]. The 5′-32P-labeled 15-/46-mer primer-template (sequences described above) was competed for extension with each unlabeled template-primer or challenge DNA (single- or double-stranded 46-mer DNA). Equimolar (10 nm) 5′-32P-labeled template-primer and unlabeled template-primer/the challenge DNA were incubated on ice for 5 min with wild type (26 nm) or G952A (140 nm) pol α in 25 μl containing 100 mm Tris-HCl, pH 8.0, 5 mm MgCl2, 50 mm KCl, 100 ng/ml BSA, 2 mm DTT. The reaction was initiated by adding 1250 μm dCTP and 418 nm trap DNA (double-stranded M13 mp2 DNA), incubated at 37 °C for 10 min and analyzed as described above. KD(rel) was determined using the average % extension values, thus S.D. values are not given, although three independent experiments were carried out. Utilization of Damaged Templates—Substrate incorporation and nucleotide selectivity assays were carried out on damaged templates as follows. A 16-mer primer (5′-CAC TGA CTG TAT GAT G-3′) was 32P-labeled at the 5′ terminus and annealed at a molar ratio of 1:2 to a 30-mer template (5′-CTC GTC AGC ATC TTC ATC ATA CAG TCA GTG-3′), containing either a cis-syn T-T dimer (39Murata T. Iwai S. Ohtsuka E. Nucleic Acids Res. 1990; 18: 7279-7286Crossref PubMed Scopus (92) Google Scholar), a (6–4) photoproduct (40Iwai S. Shimizu M. Kamiya H. Ohtsuka E. J. Am. Chem. Soc. 1996; 118: 7642-7643Crossref Scopus (101) Google Scholar), or undamaged bases at the underlined position. A 36-mer template (5′-TTG GCT GCA GAA TAT TGC TAG CGG GAA TTC GGC GCG-3′), containing either T or an abasic site (underlined position; The Midland Certified Reagent Company, Inc., Midland, TX), was annealed to a 32P-labeled 30-mer primer (5′-CGC GCC GAA TTC CCG CTA GCA ATA TTC TGC-3′). The reactions were performed at 37 °C for 60 min in a5-μl reaction containing 100 mm Tris-HCl, pH 8.0, 5 mm MgCl2,50mm KCl, 100 ng/μl BSA, 2 mm DTT, 4 nm template-primer, and 1250 μm dNTP. Kinetic parameters for single nucleotide insertion opposite the abasic site were performed at 37 °C for 30 min in a 5-μl mixture containing 100 mm Tris HCl, pH 8.0, 5 mm MgCl2, 50 mm KCl, 100 ng/μl BSA, 2 mm DTT, 40 nm template-primer, and various amounts of enzyme and dNTP. For wild type, 312–4500 μm of each nucleotide and 5.8 – 87.7 nm enzyme were used. In reactions with G952A, substrate concentration and enzyme were 39 – 4500 μm and 7.4–21 nm, respectively. Primer Extension Analysis—Gly-952 is one of the critical residues for the functions of S. cerevisiae DNA pol α (32Ogawa M. Limsirichaikul S. Niimi A. Iwai S. Yoshida S. Suzuki M. J. Biol. Chem. 2003; 278: 19071-19078Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Mutants of Gly-952 do not complement the temperature sensitivity of pol α-deficient yeast, and Ala, Tyr, Arg, and Glu substitution mutants of Gly-952 have severely impaired catalytic function. The catalytic activity of Glu and Arg substitution mutants was not measurable, and specific activity of the Tyr and Ala mutants was reduced 260- and 1500-fold, respectively. The crystal structure of RB69 DNA polymerase suggests a putative function for Gly-952 in S. cerevisiae DNA pol α. In this structure, the residue analogous to Gly-952 lies in close proximity with the template base (30Franklin M.C. Wang J. Steitz T.A. Cell. 2001; 105: 657-667Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar) (Fig. 1), suggesting that Gly-952 may interact with the template. This interaction may be altered in Gly-952 mutants leading to loss of fidelity. This possibility was tested by measuring misincorporation using a primer extension assay in the presence of the correct or incorrect nucleotide substrate. Fig. 2A shows a primer extension assay with wild type, G952A, and G952Y pol α. Wild type pol α incorporated primarily dGMP opposite template C with some misincorporation of dTMP and dAMP. In contrast, G952Y incorporated dAMP and dGMP with equal efficiency (Fig. 2A, lanes 11–12), and G952A incorporated the three incorrect nucleotides and dGMP in similar amounts (Fig. 2A, lanes 15–18). The low fidelity extension was also observed by using other three template bases; templates T, A, and G (data not shown). No incorporation was observed when the template was omitted from reactions with wild type or mutant pol α (Fig. 2A, lanes 7, 13, and 19), showing that the misincorporations are template-dependent. These results demonstrate that G952Y and G952A pol α are low fidelity DNA polymerases. Processivity Analysis—Results of the primer extension assay show that wild type pol α elongates the primer to nearly the full-length of the 46-mer template (Fig. 2A, lane 2), but Gly-952 mutants are much less efficient, adding one or two nucleotides to the primer and producing a 15- or 16-mer (Fig. 2A, lanes 8 and 14). This suggests that the mutant polymerases may have reduced processivity. The processivity was measured directly using a poly(dA) template and correct nucleotide dTTP. With limiting enzyme, wild type pol α incorporated up to 10 nucleotides, but mutant polymerases G952Y and G952A incorporated one to four nucleotides (Fig. 2B). These results suggest that G952Y and G952A have intrinsic low processivity, which contributes to low efficiency during the primer extension assay (Fig. 2A). However, short primer elongation might also result if the mutant polymerases generate mismatched primer termini that are extended poorly. The following observations support this idea. 1) In the presence of four dNTPs, primer extension products were shorter for G952A than for G952Y (Fig. 2A, lanes 8 and 14); however, these enzymes have similar processivity (Fig. 2B, lanes 9 and 17; measured on poly(dA) in the presence of dTTP such that the enzyme can not form mismatches). 2) Primer extension products tended to form a doublet (Fig. 2A, lanes 8 and 14) 2Doublet bands were observed with shorter exposure time and on the digital image from the Fuji Image Analyzer (data not shown). or to migrate at illegitimate positions (compare Fig. 2A, lanes 14 and 18). These results suggest that mutant polymerases generated the mismatched primer termini, which forced to cease the primer extension. Therefore we examined single nucleotide insertion and misextension kinetics of Gly-952 pol α mutants. Determination of Kinetic Values—Steady-state kinetic parameters for single nucleotide incorporation were determined using a gel-based assay (Table I). This assay measures the discrimination factor (DF), which is the ratio kcat/Km (i.e. incorporation efficiency) for the incorrect nucleotide to kcat/Km for the correct nucleotide. Wild type pol α incorporated the incorrect nucleotide with discrimination factors between 3.3 × 10–5 and 8.4 × 10–4 (Table I). Kinetic parameters for wild type S. cerevisiae pol α were similar to kinetic parameters of calf thymus, Drosophila, and human pol α (41Copeland W.C. Lam N.K. Wang T.S.-F. J. Biol. Chem. 1993; 268: 11041-11049Abstract Full Text PDF PubMed Google Scholar, 42Mendelman L.V. Boosalis M.S. Petruska J. Goodman M.F. J. Biol. Chem. 1989; 264: 14415-14423Abstract Full Text PDF PubMed Google Scholar, 43Perrino F.W. Loeb L.A. J. Biol. Chem. 1989; 264: 2898-2905Abstract Full Text PDF PubMed Google Scholar). However, at some template bases, the kinetics of S. cerevisiae pol α differed by >10-fold from some other α polymerases. For example, S. cerevisiae pol α incorporated A:dGMP, C:dAMP, and T:dGMP more efficiently than calf thymus pol α (43Perrino F.W. Loeb L.A. J. Biol. Chem. 1989; 264: 2898-2905Abstract Full Text PDF PubMed Google Scholar). However, this result may reflect different nucleotide sequences instead of reflecting different biochemical characteristics of different enzymes (for example see Ref. 42Mendelman L.V. Boosalis M.S. Petruska J. Goodman M.F. J. Biol. Chem. 1989; 264: 14415-14423Abstract Full Text PDF PubMed Google Scholar).Table IIncorporation kinetics by wild type, G952Y, and G952A mutantsApparent Km and kcat values were determined by Hanes-Woolf plot. The kinetic values are the average of at least triplicate determinations and are shown with S.D. Incorporation efficiency (kcat/Km) is represented as finc. DF, discrimination factor, is a relative value to kcat/Km of the correct incorporations. ND, not detectable.Template: dNMPWild typeG952YG952AKmkcatfincDFKmkcatfincDFfinc(G952Y)/finc(WT)KmkcatfincDFfinc(G952A)/finc(WT)μMmin-1μM-1 min-1μMmin-1μM-1 min-1μMmin-1μM-1 min-1T:dTMP2300 ± 200(3.6 ± 0.03) × 10-2(1.5 ± 0.1) × 10-52.1 ×10-42400 ± 100(3.6 ± 0.7) × 10-2(1.5 ± 0.2) × 10-53.2 × 10-212400 ± 5000.23 ± 0.01(9.6 ± 1.9) × 10-53.86.4T:dCMP2200 ± 300(1.0 ± 0.3) × 10-2(4.5 ± 1.9) × 10-66.2 × 10-51600 ± 400(1.1 ± 0.5) × 10-2(7.1 ± 1.8) × 10-61.5 × 10-21.5900 ± 50(8.9 ± 3.1) × 10-2(9.9 ± 4.0) × 10-53.922T:dAMP2.6 ± 0.80.19 ± 0.002(7.2 ± 1.0) × 10-211200 ± 3000.55 ± 0.06(4.6 ± 0.7) × 10-411/1602200 ± 700(5.6 ± 0.6) × 10-2(2.5 ± 0.8) × 10-511/2900T:dGMP1700 ± 2000.10 ± 0.02(6.1 ± 4.0) × 10-58.4 × 10-41000 ± 500(2.8 ± 0.4) × 10-2(2.8 ± 1.5) × 10-56.0 × 10-21/2.22100 ± 800(7.9 ± 3.0) × 10-4(3.8 ± 1.0) × 10-71.5 × 10-21/160C:dTMP2200 ± 1300(4.6 ± 1.2) × 10-2(2.1 ± 1.9) × 10-55.4 × 10-48300 ± 1500(5.9 ± 2.1) × 10-2(7.1 ± 1.8) × 10-61.5 × 10-21/33000 ± 5000.19 ± 0.07(6.3 ± 2.2) × 10-55.63C:dCMPNDNDNDND6100
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