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• W2020462817 abstract "Structural differences between class A and B DNA polymerases suggest that the motif B region, a wall of the catalytic pocket, may have evolved differentially in the two polymerase families. This study examines the function of the motif B residues in Saccharomyces cerevisiae DNA polymerase α (pol α). Effects of the mutations were determined by biochemical analysis and genetic complementation of a yeast strain carrying a temperature-sensitive pol α mutant. Many conserved residues were viable with a variety of substitutions. Among them, mutations at Asn-948 or Tyr-951 conferred up to 8-fold higher colony formation frequency in a URA3 forward mutation assay, and 79-fold higher trp1 reversion frequency was observed for Y951P in yeast. Purified Y951P was as accurate as wild type in DNA synthesis but ∼6-fold less processive and 22-fold less active in vitro. Therefore, Y951P may increase the frequency of mutant colony formation because of its low level of DNA polymerase activity in yeast. Mutations at Lys-944 or Gly-952 were not viable, which is consistent with the observation that mutants with substitutions at Gly-952 have strongly reduced catalytic activity in vitro. Gly-952 may provide a space for the nascent base pair and thus may play an essential function in S. cerevisiae DNA pol α. These results suggest that class B DNA polymerases have a unique structure in the catalytic pocket, which is distinct from the corresponding region in class A DNA polymerases. Structural differences between class A and B DNA polymerases suggest that the motif B region, a wall of the catalytic pocket, may have evolved differentially in the two polymerase families. This study examines the function of the motif B residues in Saccharomyces cerevisiae DNA polymerase α (pol α). Effects of the mutations were determined by biochemical analysis and genetic complementation of a yeast strain carrying a temperature-sensitive pol α mutant. Many conserved residues were viable with a variety of substitutions. Among them, mutations at Asn-948 or Tyr-951 conferred up to 8-fold higher colony formation frequency in a URA3 forward mutation assay, and 79-fold higher trp1 reversion frequency was observed for Y951P in yeast. Purified Y951P was as accurate as wild type in DNA synthesis but ∼6-fold less processive and 22-fold less active in vitro. Therefore, Y951P may increase the frequency of mutant colony formation because of its low level of DNA polymerase activity in yeast. Mutations at Lys-944 or Gly-952 were not viable, which is consistent with the observation that mutants with substitutions at Gly-952 have strongly reduced catalytic activity in vitro. Gly-952 may provide a space for the nascent base pair and thus may play an essential function in S. cerevisiae DNA pol α. These results suggest that class B DNA polymerases have a unique structure in the catalytic pocket, which is distinct from the corresponding region in class A DNA polymerases. During cell division, a mother cell divides to generate two identical daughter cells, each of which receives a complete genetic complement. DNA replication is a critical step, which duplicates the parental chromosomes prior to cell division in the cell cycle. In eukaryotic cells, at least three DNA polymerases participate in replication of nuclear DNA: DNA polymerases α (pol α), 1The abbreviations used are: pol, DNA polymerase; FOA, fluoroorotic acid; BSA, bovine serum albumin; DTT, dithiothreitol. δ, and ϵ, all of which belong to class B (pol α) DNA polymerases (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 (284) Google Scholar). The pol α/DNA primase complex plays a key role in the initiation phase of DNA replication; pol δ and pol ϵ play roles primarily in the elongation phase. During initiation of DNA synthesis, DNA primase synthesizes a short RNA chain (∼10-mer), and pol α extends the RNA primer by ∼30 deoxyribonucleotides. Pol ϵ and pol δ are highly processive DNA polymerases that carry out bidirectional DNA synthesis during the elongation phase of DNA replication (2Waga S. Stillman B. Annu. Rev. Biochem. 1998; 67: 721-751Crossref PubMed Scopus (663) Google Scholar). Class B family DNA polymerases share structures and catalytic mechanism with class A DNA polymerases such as Taq DNA pol I (3Steitz T.A. J. Biol. Chem. 1999; 274: 17395-17398Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar, 4Patel P.H. Loeb L.A. Nat. Struct. Biol. 2001; 8: 656-659Crossref PubMed Scopus (77) Google Scholar). Motif B is an essential region conserved in class A DNA polymerases. A Lys residue is essential in motif B in Taq pol I (Lys-663) and Klenow fragment (Lys-758) of Escherichia coli pol I (5Astatke 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, 6Pandey V.N. Kaushik N. Modak M.J. J. Biol. Chem. 1994; 269: 13259-13265Abstract Full Text PDF PubMed Google Scholar, 7Suzuki M. Baskin D. Hood L. Loeb L.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9670-9675Crossref PubMed Scopus (65) Google Scholar). Phe-667 in Taq (and the equivalent residues in Klenow and T7 pol) are also critical for polymerase activity, as well as discrimination of 2′,3′-dideoxyribonucleotides (5Astatke 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, 8Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6339-6343Crossref PubMed Scopus (302) Google Scholar, 9Astatke M. Grindley N.D. Joyce C.M. J. Mol. Biol. 1998; 278: 147-165Crossref PubMed Scopus (99) Google Scholar). Tyr-766 in Klenow fragment (Tyr-671 in Taq pol I) plays a role in fidelity of DNA synthesis (10Bell 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, 11Carroll S.S. Cowart M. Benkovic S.J. Biochemistry. 1991; 30: 804-813Crossref PubMed Scopus (116) Google Scholar). Recently, Ponamarev et al. (12Ponamarev 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 (115) 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. 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 (13Li Y. Korolev S. Waksman G. EMBO J. 1998; 17: 7514-7525Crossref PubMed Scopus (659) Google Scholar, 14Doublie S. Tabor S. Long A.M. Richardson C.C. Ellenberger T. Nature. 1998; 391: 251-258Crossref PubMed Scopus (1105) Google Scholar, 15Kiefer J.R. Mao C. Braman J.C. Beese L.S. Nature. 1998; 391: 304-307Crossref PubMed Scopus (483) Google Scholar, 16Suzuki 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). Lys-663, Phe-667, and Tyr-671 in Taq pol I (and the corresponding residues in other class A DNA polymerases) and Arg-659 in Taq pol I are essential for genetic complementation of a pol Its strain of E. coli (7Suzuki M. Baskin D. Hood L. Loeb L.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9670-9675Crossref PubMed Scopus (65) Google Scholar). These motif B residues may constitute the wall of the catalytic pocket together with residues located in other motifs (for example see Refs. 17Minnick D.T. Bebenek K. Osheroff W.P. Turner Jr., R. 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, 18Patel P.H. Loeb L.A. J. Biol. Chem. 2000; 275: 40266-40272Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 19Patel P.H. Loeb L.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5095-5100Crossref PubMed Scopus (86) Google Scholar, 20Patel P.H. Suzuki M. Adman E. Shinkai A. Loeb L.A. J. Mol. Biol. 2001; 308: 823-837Crossref PubMed Scopus (160) Google Scholar, 21Shinkai A. Patel P.H. Loeb L.A. J. Biol. Chem. 2001; 276: 18836-18842Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 22Shinkai A. Loeb L.A. J. Biol. Chem. 2001; 276: 46759-46764Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Some of the motif B residues are also conserved in DNA polymerases of other classes. The comparable residues in class B polymerases are located in the fingers subdomain and may face toward the polymerase cleft (23Hopfner K.P. Eichinger A. Engh R.A. Laue F. Ankenbauer W. Huber R. Angerer B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3600-3605Crossref PubMed Scopus (195) Google Scholar, 24Rodriguez A.C. Park H.W. Mao C. Beese L.S. J. Mol. Biol. 2000; 299: 447-462Crossref PubMed Scopus (120) Google Scholar, 25Wang J. Sattar A.K. Wang C.C. Karam J.D. Konigsberg W.H. Steitz T.A. Cell. 1997; 89: 1087-1099Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar, 26Zhao Y. Jeruzalmi D. Moarefi I. Leighton L. Lasken R. Kuriyan J. Struct. Fold Des. 1999; 7: 1189-1199Abstract Full Text Full Text PDF Scopus (87) Google Scholar). Mutations of the essential Lys greatly reduce polymerase activity in human pol α, ϕ29 pol, RB69 pol (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. Lin T. Karam J. Konigsberg W.H. Biochemistry. 1999; 38: 8094-8101Crossref PubMed Scopus (44) Google Scholar), and Klenow fragment (5Astatke 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, 6Pandey V.N. Kaushik N. Modak M.J. J. Biol. Chem. 1994; 269: 13259-13265Abstract Full Text PDF PubMed Google Scholar). Therefore, this motif B Lys may be functionally conserved in class A and B DNA polymerases. In Taq pol I, other motif B residues also play a role in fidelity of DNA synthesis (16Suzuki 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, 30Suzuki 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, 31Tosaka A. Ogawa M. Yoshida S. Suzuki M. J. Biol. Chem. 2001; 276: 27562-27567Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 32Yoshida 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), although less function is known about the other motif B residues of class B DNA polymerases. This study analyzes the role of motif B residues in Saccharomyces cerevisiae pol α. Motif B mutants were created using randomized cassette mutagenesis and screened for genetic complementation of temperature-sensitive pol1–17. Specific mutants of interest were purified and characterized. The results are discussed in terms of the structure-function relationships in DNA polymerase motif B. Strains, Plasmids, and Oligonucleotides—Yeast strain S111pol1–17, trp1–289 tyr1 ura3–1 ura3–2 ade2–101 gal2 can1 pol1–17 (33Budd M. Campbell J.L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2838-2842Crossref PubMed Scopus (69) Google Scholar), and YCplac33, a genomic clone of S. cerevisiae pol α (34Gietz R.D. Sugino A. Gene. 1988; 74: 527-534Crossref PubMed Scopus (2522) Google Scholar), were generously provided by Hiroyuki Araki at the National Institute of Genetics. Vector Construction—Mutant cassettes were inserted into unique restriction sites upstream and downstream of the motif B region (see Fig. 1). The restriction sites were created using the GeneEditor in vitro site-directed mutagenesis kit (Promega). Oligonucleotides with silent mutations (underlined) were as follows: +SacII, 5′-T CAG GGT GTT TTA CCGCGG TTA TTA GCT CTG; +XhoI, 5′-TTG GGT TAT GTT AAC TCG AGA TTT TAC GCA AAG C; -XhoI, 5′-GAT CCA CAA TAT TAT CTG GAG AAA CAA ATA TTC G. In this protocol, the +SacII and the +XhoI primers produced new restriction sites, and the -XhoI primer destroyed an XhoI site farther downstream. Prior to random library construction, the SacII-XhoI fragment was replaced with the sequence that has a stop codon (TGA) in place of Lys-944 (non-functional vector). Preparation of Motif B Cassettes with Randomized Sequence—Random mutagenesis was performed essentially as described (7Suzuki M. Baskin D. Hood L. Loeb L.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9670-9675Crossref PubMed Scopus (65) Google Scholar, 19Patel P.H. Loeb L.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5095-5100Crossref PubMed Scopus (86) Google Scholar, 21Shinkai A. Patel P.H. Loeb L.A. J. Biol. Chem. 2001; 276: 18836-18842Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 35Glick E. Vigna K.L. Loeb L.A. EMBO J. 2001; 20: 7303-7312Crossref PubMed Scopus (43) Google Scholar, 36Kim B. Hathaway T.R. Loeb L.A. J. Biol. Chem. 1996; 271: 4872-4878Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Oligonucleotides containing randomized sequence were synthesized by Integrated DNA Technologies (Commercial Park, Coralville, IA) as follows: 6% RANDOM (antisense strand), 5′-TT TGC GTA AAA TCT CGA GTT [AAC ATA ACC CAA ACA ACC ATA CAT AGA ATT GGC AGT CAA TTT TAG AGC TTG TTG ACG AAT ATC ACA TTG] AAC TCG CTT ATG GGG ATC AG, in which each nucleotide in brackets was 94% wild type nucleotide and 2% each of the other three nucleotides; 6% RANDOM (sense strand), 5′-GGG TGT TTT CCG CGG TTA TTA GCT AAT CTG GTG GAT CGT CGA CGT GAA GTT AAG AAG GTG ATG AAA ACT GAA ACT GAT CCC CAT AAG CGA GTT, in which 20 3′-terminal nucleotides are complementary to the 3′-terminal nucleotides of the 6% RANDOM (antisense strand). 25 cycles of PCR were carried out in a reaction containing 50 mm Tris-HCl, pH 8.0, 2 mm MgCl2,50 μm each of dNTP, 50 mm KCl, 2.5 units of Taq pol I, each 0.02 pmol of 6% RANDOM (sense strand) and 6% RANDOM (antisense strand) oligomers, and 10 pmol each of two primers (amp-top, GGG TGT TTT ACC GCG GTT AT; and amp-bottom, TTT GCG TAA AAT CTC GAG TT). The PCR product (6% random fragment) contains on average three amino acid substitutions (37Suzuki M. Christians F.C. Kim B. Skandalis A. Black M.E. Loeb L.A. Mol. Divers. 1996; 2: 111-118Crossref PubMed Scopus (27) Google Scholar). For totally randomized libraries, oligonucleotides that contain an equal mixture of the four nucleotides at Gln-940, Lys-944, Asn-948, Tyr-951, or Gly-952 were prepared, and each fragment was amplified by the same procedure as 6% random fragment (totally randomized fragments). Library Construction—Non-functional vector and 6% random or totally randomized fragments were digested with SacII and XhoI, purified, mixed, and ligated by T4 DNA ligase. Constructs were transformed into DH5α cells. An aliquot was spread over a solid LB agar plate containing 100 μg/ml carbenicillin, and the rest were cultured in 5 ml of liquid medium. After the cells were cultured for 16 h, library size was estimated by counting transformants. Twelve sample clones from each library were subcultured and sequenced to confirm that substitutions were introduced randomly. All libraries contained various types of substitutions at the specified codons (data not shown). Plasmids were purified from liquid culture (6% random library plasmid or totally randomized library plasmid) and transformed into yeast pol1–17 (see below). The plasmid propagates at a low copy number in yeast, producing a low level of transcription of the cloned mutant DNA polymerase, expected to be similar to the level at which the endogenous DNA polymerase is expressed. The 6% random library includes ∼45,000 independent clones with an average nucleotide substitution number of 4.1. Complementation Assay—The 6% random library plasmid was introduced into the pol1–17 strain using Frozen-EZ Yeast Transformation II (ZYMO RESEARCH, Orange, CA). Transformants were cultured on SD-URA solid plates (6.7 g/liter yeast nitrogen base without amino acids, 5 g/liter casamino acids, 20 g/liter glucose, 20 mg/liter adenine sulfate, 20 mg/liter tryptophan, 15 g/liter Bacto-agar) at 25 °C for 48 h. Replica colonies were transferred to new plates using Replica Plater (TaKaRa, Kyoto, Japan) and cultured at 37 °C for 72 h. Vector construction and complementation assay are illustrated schematically in Fig. 2. Plasmid Isolation and DNA Sequencing—Yeast colonies were picked and cultured in 2 ml of SD-URA at 37 °C overnight. Cells were collected, suspended in 500 μl of reaction buffer (1 m d-glucitol, 50 mm Tris-HCl, pH 7.5, 100 mm EDTA), and incubated with 20 μl of 10 mg/ml zymolyase-100T (SEIKAGAKUKOGYO, Tokyo, Japan) at 37 °C for 30 min with shaking. After buffer was replaced with 500 μl of TE (10 mm Tris-HCl, pH 8.0, 1 mm EDTA), cells were lysed with 50 μl of 10% SDS at 65 °C for 15 min and incubated on ice with 200 μl of 5 m potassium acetate buffer for 30 min. After centrifugation, supernatants were recovered, and proteins were removed by extraction with Phenol CIA (phenol:chloroform:isoamyl alcohol = 25:24:1, mixed pH 6.7; Nacalai Tesque, Kyoto, Japan). Plasmid DNA was precipitated with isopropanol incubated with 50 μl of TE and 1 μg/ml RNase at 37 °C for 30 min, purified by GENECLEAN II (BIO101; Vista, CA), and used to transform JM109 competent cells. Colonies were picked and inoculated into 2 ml each of 2× YT containing carbenicillin (100 μg/ml) at 37 °C overnight. Plasmids were purified, and target regions were sequenced using the Taq Dye Terminator cycle sequence kit, with a 373A DNA sequencer (Applied Biosystems). An oligomer, 5′-CAG CTA GTT GTC TTG TAT TC, was used for the sequencing primer. URA3 Forward Mutation Assay—Yeast strain pol1–17 was freshly transformed by each pol α mutant and grown on SD-Ura plates at 25 °C for 4 days. Transformants were inoculated in YPD (1% yeast extract, 2% peptone, and 2% glucose) and grown at 37 °C for 24 h. Cells were collected, washed with water, and plated onto YPD plates at ∼103 cells/plate or on YPD plates containing 0.5 mg/ml 5-fluoroorotic acid (5-FOA) at ∼107 cells/plate. After 4 days of culture at 37 °C, the number of FOA-resistant colonies was determined. Total cell number and mutation frequency per 107 cells was calculated. To analyze the mutation spectrum, the ura3 plasmid was amplified from FOA-resistant colonies. PCR primer sequences were uraT, 5′-TCC ATG CAG TTG GAC GAT CG; and uraB, 5′-AAA TAG GCG TAT CAC GAG GC. Purified PCR fragments were sequenced as described above using primers uraS1, 5′-AGT ATT CTT AAC CCA ACT GC; uraS2, 5′-TGT GCT TCA TTG GAT GTT CG; uraS3, 5′-CTT TAG CGG CTT AAC TGT GC; and uraS4, 5′-TAA CAA AGG AAC CTA GAG GC. trp1 Reversion Assay—trp1 reversion assay was performed using essentially the same procedure as the 5-FOA forward mutation assay, except that cells were plated on SD-URA-TRP plates (6.7 g/liter yeast nitrogen base without amino acids, 5 g/liter casamino acids, 20 g/liter glucose, 20 mg/liter adenine sulfate, 15 g/liter Bacto-agar). Expression and Purification of DNA Polymerases—A fragment coding wild type was amplified by PCR, using PCR primers that carry unique restriction sites BamHI or SphI. The fragment was digested and inserted into pFastBacHTb expression vector (pFastBac-Wt; Invitrogen). Y951P, G952R, and G952E were obtained from random library vector. G952Y and G952A mutants were constructed by cassette mutagenesis. Cassette fragments were amplified using the sense oligomer, 5′-GGGTGTTTTACCAAGGTTAT-3′, and one of two antisense oligomers, 5′-TTT GCG TAA AAT CTC GAG TTA ACA TAA CCC AAA CAAGCA T AC ATA GAA TT G GCA G, for Ala substitution, and the same sequence except ATA (underlined positions) for Tyr substitution. Mutants were constructed by inserting the mutant cassette into XhoI and SacII sites of the YCplac33 shuttle vector carried wild type pol α. Open reading frames carrying the mutations were confirmed by DNA sequencing and subcloned into MluI and XhoI sites of pFastBacHTb-Wt histidine-tagged pol α, and its derivatives were expressed and purified using the BAC-TO-BAC HT baculovirus expression system (Invitrogen). E. coli DH10bac (Invitrogen) was transformed with pFastBac HTb vectors. A single colony containing the recombinant bacmid was picked, inoculated into 2 ml of 2×YT, and cultured at 37 °C overnight. The bacmid DNA was purified and transfected into Sf9 cells according to the supplier's instructions (Invitrogen). At 72 h post-infection, cells (2.5 × 107) were harvested by centrifugation at 500 × g, lysed, and homogenized in lysis buffer (50 mm Tris-HCl, pH 8.0, 100 mm KCl, 20 mm imidazole, 5 mm 2-mercaptoethanol, 1 mm phenylmethylsulfonyl fluoride, 1% Nonidet P-40) at 4 °C. The lysate was incubated on ice for 1 h and centrifuged at 25,000 × g for 10 min. The supernatant was loaded onto a nickel-resin column (His-Bind Resin WI; Novagen) equilibrated with Buffer A (20 mm Tris-HCl, pH 8.0, 500 mm KCl, 20 mm imidazole, 5 mm 2-mercaptoethanol, 10% glycerol). The column was washed sequentially with 10 ml of Buffer A, 2 ml of Buffer B (20 mm Tris-HCl, pH 8.0, 1 m KCl, 5 mm 2-mercaptoethanol, 10% glycerol), and 2 ml of Buffer A. Enzyme was eluted with 5 ml of elution buffer (20 mm Tris-HCl, pH 8.0, 100 mm KCl, 100 mm imidazole, 5 mm 2-mercaptoethanol, 10% glycerol), and 0.5-ml fractions were collected. Peak fractions were dialyzed against 500 ml of dialysis buffer I (30% glycerol, 50 mm Tris-HCl, pH 8.0, 2 mm 2-mercaptoethanol) for 4 h followed by dialysis buffer II (50% glycerol, 50 mm Tris-HCl, pH 8.0, 2 mm 2-mercaptoethanol) for 4 h. Purity was checked by SDS-PAGE. Protein concentration was determined using Bradford reagent (Bio-Rad). Primer Extension Assay—Primer extension assay was described previously (30Suzuki 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). For primer extension assays on damaged DNA, a 5′-32P-labeled 15-nucleotide oligomer (5′-CAC TGA CTG TAT GAT) was annealed to the 30-mer template (5′ CTC GTC AGC ATC TTC ATC ATA CAG TCA GTG 3′), containing either a cis-syn T-T dimer (38Murata T. Iwai S. Ohtsuka E. Nucleic Acids Res. 1990; 18: 7279-7286Crossref PubMed Scopus (92) Google Scholar) or undamaged at the underlined position, at a molar ratio 1:2. A 36-mer template (5′ TTG GCT GCA GAA TAT TGC TAG CGG GAA TTC GGC GCG-3′), containing T, Etheno A, abasic site, O6-methylguanine, O4-methylthymine, or 8-hydroxyguanine at the underlined position (The Midland Certified Reagent Company, Inc., Midland, TX) was prepared by annealing a 32P-labeled 28-mer primer (5′-CGC GCC GAA TTC CCG CTA GCA ATA TTC T) at molar ratio of 1:2. The reactions were performed at 37 °C for 60 min in 20 μl containing 2 mm MgCl2, 50 mm Tris-HCl, pH 8.0, 50 mm KCl, 100 ng/μl BSA, 2 mm DTT, 33 nm pol α, 4 nm32P-labeled primer-template DNA, and 100 μm dNTP. Reactions were terminated by addition of an equal volume of 2× loading buffer containing 90% formamide, 20 mm EDTA, 0.05% xylene cyanol, and 0.05% bromphenol blue. Reaction products were analyzed by 14% polyacrylamide gel electrophoresis. Processivity Assay—An oligo(dT)16-oligomer (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 25 μl containing 100 mm Tris-HCl, pH 8.0, 5 mm MgCl2, 50 mm KCl, 100 ng/μl BSA, 2 mm DTT, 300 μm dTTP, 40 ng/μl template annealed to 4 ng/μl primer; enzyme concentration was varied in range of 0.35 to 180 nm 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 polyacrylamide gel electrophoresis. Specific Activity—Polymerase activity was assayed in a 25-μl reaction containing 80 mm potassium phosphate, pH 7.2, 8 mm 2-mercaptoethanol, 200 μg/ml of activated calf thymus DNA, 80 μm each of dATP, dGTP, and dCTP, 40 μm each of dTTP and [3H]dTTP (18.5 kBq), and 8 mm MgCl2. After incubation at 37 °C for 60 min, acid-insoluble radio-activity was measured as described previously (39Yoshida S. Kondo T. Ando T. Biochim. Biophys. Acta. 1974; 353: 463-474Crossref PubMed Scopus (98) Google Scholar). One unit represents incorporation of 1 nmol of dNMP in 1 h. Wild type pol α had a specific activity of 2.8 × 104 units/mg. Single Nucleotide Incorporation Kinetics—Steady-state kinetic constants were determined for incorporation of dGMP (Ultrapure dNTP; Amersham Biosciences). 32P-Labeled 14-mer (5′-CGC GCC GAA TTC CC) primer was annealed to 46-mer of oligonucleotide, 5′-GCG CGG AAG CTT GGC TGC AGA ATA TTG CTA GCG GGA ATT CGG CGC G-3′, where underlined C was the target template site. Reaction conditions were used such that 20% of template-primer was utilized (40Goodman M.F. Creighton S. Bloom L.B. Petruska J. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 83-126Crossref PubMed Scopus (403) 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 min (wild type and Y951P) 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. Apparent kinetic parameters (Km and kcat) for incorporation of dGMP was determined by Hanes-Woolf plots. 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 values were determined with DNA concentrations of 40 or 200 nm, using the equation, KD = [Dlow](kcat/Km)high/(kcat/Km)low (41Creighton 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 to measure the ratio of polymerase bound to template primer termini and to single-stranded or double-stranded challenge DNA. First, an experiment is done with equimolar labeled and unlabeled template-primer DNA. The fraction of the initial labeled primer extended by the polymerase is quantified (i.e. % extension(template-primer dna)). In the second experiment, extension of the labeled primer-template DNA in the presence of equimolar challenge DNA (single- or double-stranded DNA) is quantified. Fraction of the extended primer DNA is denoted as % extension(challenge dna). The KD(rel) value for each template combination is calculated using the following equation, KD(rel) = KD(challenge dna)/KD(template-primer dna) = % extension(challenge dna)/[2 × % extension(template-primer dna) – % extension(challenge dna)] (41Creighton S. Huang M.M. Cai H. Arnheim N. Goodman M.F. J. Biol. Chem. 1992; 267: 2633-2639Abstract Full Text PDF PubMed Google Scholar). The 5′-32P labeled 14-/46-mer primer-template (described above) was competed for extension with each template-primer or challenge DNA (single- or double-stranded 46-mer DNA). Equimolar (10 nm) 5′-32P-labeled template-primer and the template-primer/challenge DNA were incubated on ice for 5 min with wild type (30 nm) or Y951P (48 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 300 μm dGTP and 418 nm trap DNA (double-stranded M13 mp2 DNA) and incubated at 37 °C for 5 min. In the formula, KD(rel) was determined using average % extension values, thus S.D. values are not given, although three independent experiments were carried out. Vector Construction, Genetic Complementation, and Purification of Taq pol I—To randomize Gly-672 of Taq pol I, oligonucleotides containing totally random sequence in which N represents a mixture of four dNMP were synthesized: O+GN RANDOM, 5′-CGG GAG GCC GTG GAC CCC CTG ATG CGC CGG GCG GCC AAG ACC ATC AAC TTC GGG GTC CTC TAC NNN ATG TCG GCC CAC CG; O-0G primer, 5′-GTA AGG GAT GGC TAG CTC CTG GGA GAG GCG GTG GGC CGA CAT; O(+)G primer, 5′-TTC GGC GTC CCG CGG GAG GCC GTG GAC CCC CTG. The library was constructed and screened for genetic complementation in pol Its strain in E. coli as described previously (7Suzuki M. Baskin D. Hood L. Loeb L.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9670-9675Crossref PubMed Scopus (65) Google Scholar). Selection of Functional Mutants from 6% Random Library— Motif B is a conserved motif in class A and B DNA polymerases that forms a wall in the fingers domain and is part of the catalytic pocket (13Li Y. Korolev S. Waksman G. EMBO J. 1998; 17: 7514-7525Crossref PubMed Scopus (659) Google Scholar, 42Franklin M.C. Wang J. Steitz T.A. Cell. 2001; 105: 657-667Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar). The fingers domain includes residues that perform essential functions during enzyme catalysis; however, the ternary structure of motif B varies in different DNA polymerases. For example, in Taq pol I (class A) the single O-helix interacts with the substrate. In RB69 DNA polymerase (class B), the helix P may play the comparable roles, but the second helix N may also be employed for" @default.
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• W2020462817 title "Distinct Function of Conserved Amino Acids in the Fingers of Saccharomyces cerevisiae DNA Polymerase α" @default.
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