The (I/Y)XGG Motif of Adenovirus DNA Polymerase Affects Template DNA Binding and the Transition from Initiation to Elongation
2001; Elsevier BV; Volume: 276; Issue: 32 Linguagem: Inglês
10.1074/jbc.m103159200
ISSN1083-351X
AutoresArjan B. Brenkman, Marinus R. Heideman, Verónica Truniger, Margarita Salas, Peter C. van der Vliet,
Tópico(s)Cytomegalovirus and herpesvirus research
ResumoAdenovirus DNA polymerase (Ad pol) is a eukaryotic-type DNA polymerase involved in the catalysis of protein-primed initiation as well as DNA polymerization. The functional significance of the (I/Y)XGG motif, highly conserved among eukaryotic-type DNA polymerases, was analyzed in Ad pol by site-directed mutagenesis of four conserved amino acids. All mutant polymerases could bind primer-template DNA efficiently but were impaired in binding duplex DNA. Three mutant polymerases required higher nucleotide concentrations for effective polymerization and showed higher exonuclease activity on double-stranded DNA. These observations suggest a local destabilization of DNA substrate at the polymerase active site. In agreement with this, the mutant polymerases showed reduced initiation activity and increasedK m(app) for the initiating nucleotide, dCMP. Interestingly, one mutant polymerase, while capable of elongating on the primer-template DNA, failed to elongate after protein priming. Further investigation of this mutant polymerase showed that polymerization activity decreased after each polymerization step and ceased completely after formation of the precursor terminal protein-trinucleotide (pTP-CAT) initiation intermediate. Our results suggest that residues in the conserved motif (I/Y)XGG in Ad pol are involved in binding the template strand in the polymerase active site and play an important role in the transition from initiation to elongation. Adenovirus DNA polymerase (Ad pol) is a eukaryotic-type DNA polymerase involved in the catalysis of protein-primed initiation as well as DNA polymerization. The functional significance of the (I/Y)XGG motif, highly conserved among eukaryotic-type DNA polymerases, was analyzed in Ad pol by site-directed mutagenesis of four conserved amino acids. All mutant polymerases could bind primer-template DNA efficiently but were impaired in binding duplex DNA. Three mutant polymerases required higher nucleotide concentrations for effective polymerization and showed higher exonuclease activity on double-stranded DNA. These observations suggest a local destabilization of DNA substrate at the polymerase active site. In agreement with this, the mutant polymerases showed reduced initiation activity and increasedK m(app) for the initiating nucleotide, dCMP. Interestingly, one mutant polymerase, while capable of elongating on the primer-template DNA, failed to elongate after protein priming. Further investigation of this mutant polymerase showed that polymerization activity decreased after each polymerization step and ceased completely after formation of the precursor terminal protein-trinucleotide (pTP-CAT) initiation intermediate. Our results suggest that residues in the conserved motif (I/Y)XGG in Ad pol are involved in binding the template strand in the polymerase active site and play an important role in the transition from initiation to elongation. terminal protein adenovirus polymerase exonuclease precursor terminal protein single-stranded DNA double-stranded DNA dithiothreitol bovine serum albumin Adenoviruses contain a linear double-stranded genome of ∼36 kilobases with two origins of replication located in the inverted terminal repeats. At each 5′-end of the adenovirus genome, a terminal protein (TP)1 is covalently linked. Replication initiates via a protein-priming mechanism (1Salas M. Annu. Rev. Biochem. 1991; 60: 39-71Crossref PubMed Scopus (347) Google Scholar) involving the Ad pol and precursor terminal protein (pTP). Ad pol and pTP form a tight heterodimer of which the pTP acts as a primer and is covalently linked to the initiating nucleotide dCMP. Initiation of replication is catalyzed by Ad pol and can be stimulated by the two cellular transcription factors NFI and Oct-1, which function to recruit and position the pTP-pol complex on the origin of replication (Ref. 2de Jong R.N. van der Vliet P.C. Gene (Amst.). 1999; 236: 1-12Crossref PubMed Scopus (67) Google Scholar and references therein). Ad pol initiates replication opposite the fourth base of the template strand and synthesizes a pTP-CAT intermediate. For elongation to occur, this intermediate jumps back to be paired with template residues 1–3, after which pTP dissociates from Ad pol and elongation starts (3King A.J. van der Vliet P.C. EMBO J. 1994; 13: 5786-5792Crossref PubMed Scopus (73) Google Scholar, 4King A.J. Teertstra W.R. van der Vliet P.C. J. Biol. Chem. 1997; 272: 24617-24623Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Elongation occurs via a strand displacement mechanism that requires the viral DNA-binding protein (reviewed in Refs. 5Hay R.T. Adenovirus DNA Replication.in: DePamphilis M.L. DNA Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996Google Scholar and 6van der Vliet P.C. Curr. Top. Microbiol. Immunol. 1995; 199: 1-30PubMed Google Scholar). Late in infection, pTP is cleaved by a virus-encoded protease into TP and the precursor part (reviewed in Refs. 5Hay R.T. Adenovirus DNA Replication.in: DePamphilis M.L. DNA Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996Google Scholar and 6van der Vliet P.C. Curr. Top. Microbiol. Immunol. 1995; 199: 1-30PubMed Google Scholar). The actual role of pTP processing is at present unclear. Initiation and elongation are performed by the same polymerase, but the two processes differ in sensitivity to inhibitors (7Nagata K. Guggenheimer R.A. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4266-4270Crossref PubMed Scopus (80) Google Scholar, 8Sussenbach J.S. van der Vliet P.C. Curr. Top. Microbiol. Immunol. 1984; 109: 53-73PubMed Google Scholar). This suggests that a conformational change occurs upon transition from initiation to elongation, most likely after the formation of pTP-CAT. In agreement with this notion, kinetic studies revealed that the K m for dCTP is lower for initiation than for elongation (9Mul Y.M. van der Vliet P.C. Nucleic Acids Res. 1993; 21: 641-647Crossref PubMed Scopus (34) Google Scholar). In addition to its synthetic activities, Ad pol also possesses a distributive 3′–5′-exonuclease activity, shown to be involved in proofreading (10King A.J. Teertstra W.R. Blanco L. Salas M. van der Vliet P.C. Nucleic Acids Res. 1997; 25: 1745-1752Crossref PubMed Scopus (25) Google Scholar). Many DNA polymerases have been characterized and were generally found to have a polymerase and a 3′–5′-exonuclease activity. Sequence comparisons of DNA polymerases from bacterial, viral, and cellular origin led to a classification into four groups, A, B (also known as α-like), C, and D, based on amino acid similarities withEscherichia coli pol I, II, and III, and human DNA pol β (11Braithwaite D.K. Ito J. Nucleic Acids Res. 1993; 21: 787-802Crossref PubMed Scopus (531) Google Scholar, 12Delarue M. Poch O. Tordo N. Moras D. Argos P. Protein Eng. 1990; 3: 461-467Crossref PubMed Scopus (580) Google Scholar, 13Ito J. Braithwaite D.K. Nucleic Acids Res. 1991; 19: 4045-4057Crossref PubMed Scopus (286) Google Scholar). Based on their extent of similarity, six highly conserved motifs (I–VI), which were proposed to lie in the polymerase active site, were identified in human pol α (14Wong S.W. Wahl A.F. Yuan P.M. Arai N. Pearson B.E. Arai K. Korn D. Hunkapiller M.W. Wang T.S. EMBO J. 1988; 7: 37-47Crossref PubMed Scopus (307) Google Scholar). DNA polymerases containing these six motifs (see Fig. 1) were designated α-like DNA polymerases (12Delarue M. Poch O. Tordo N. Moras D. Argos P. Protein Eng. 1990; 3: 461-467Crossref PubMed Scopus (580) Google Scholar). Further alignments showed that motifs I–III are conserved in all groups of DNA polymerases (12Delarue M. Poch O. Tordo N. Moras D. Argos P. Protein Eng. 1990; 3: 461-467Crossref PubMed Scopus (580) Google Scholar), while a seventh conserved motif was identified in the α-like DNA polymerases (15Hwang C.B. Ruffner K.L. Coen D.M. J. Virol. 1992; 66: 1774-1776Crossref PubMed Google Scholar, 16Blasco M.A. Mendez J. Lazaro J.M. Blanco L. Salas M. J. Biol. Chem. 1995; 270: 2735-2740Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Besides conserved motifs located in the C-terminal part of DNA polymerases, three sequence motifs (Exo I–III) were shown to form an evolutionary conserved 3′–5′-exonuclease site (17Blanco L. Bernad A. Salas M. Gene (Amst.). 1992; 112: 139-144Crossref PubMed Scopus (73) Google Scholar, 18Blanco L. Bernad A. Blasco M.A. Salas M. Gene (Amst.). 1991; 100: 27-38Crossref PubMed Scopus (192) Google Scholar, 19Bernad A. Blanco L. Lazaro J.M. Martin G. Salas M. Cell. 1989; 59: 219-228Abstract Full Text PDF PubMed Scopus (340) Google Scholar). Extensive biochemical analysis of a number of prokaryotic and eukaryotic DNA polymerases, such as the Klenow fragment of E. coli pol I (12Delarue M. Poch O. Tordo N. Moras D. Argos P. Protein Eng. 1990; 3: 461-467Crossref PubMed Scopus (580) Google Scholar), T4 (reviewed in Ref. 20Reha-Krantz L.J. Methods Enzymol. 1995; 262: 323-331Crossref PubMed Scopus (18) Google Scholar), herpes simplex virus (21Larder B.A. Kemp S.D. Darby G. EMBO J. 1987; 6: 169-175Crossref PubMed Scopus (163) Google Scholar), φ29 (reviewed in Ref. 22Blanco L. Salas M. Methods Enzymol. 1995; 262: 283-294Crossref PubMed Scopus (38) Google Scholar), and pol α (23Copeland W.C. Dong Q. 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Wilson S.H. Kraut J. Biochemistry. 1996; 35: 12742-12761Crossref PubMed Scopus (274) Google Scholar). They are all proposed to utilize an identical two-metal ion-catalyzed polymerase mechanism but differ extensively in many of their structural features (29Steitz T.A. J. Biol. Chem. 1999; 274: 17395-17398Abstract Full Text Full Text PDF PubMed Scopus (699) Google Scholar). The crystal structure of phage RB69 DNA polymerase (24Wang 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) can serve as prototype of the pol α family of DNA polymerases, since the recently solved crystal structures ofThermococcus gorgonarius DNA polymerase (30Hopfner 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 (196) Google Scholar) andThermococcus sp. 9 degrees N-7 (31Rodriguez A.C. Park H.W. Mao C. Beese L.S. J. Mol. Biol. 2000; 299: 447-462Crossref PubMed Scopus (120) Google Scholar) are topologically similar to this DNA polymerase. Ad pol is an α-like DNA polymerase belonging to the subclass of protein-priming DNA polymerases. Site-directed mutagenesis studies have identified motif I as a motif important for initiation and elongation activity of Ad pol (32Joung I. Horwitz M.S. Engler J.A. Virology. 1991; 184: 235-241Crossref PubMed Scopus (25) Google Scholar). Furthermore, two putative zinc finger domains were identified (33Joung I. Engler J.A. J. Virol. 1992; 66: 5788-5796Crossref PubMed Google Scholar), and linker mutagenesis studies have shown that multiple regions, including motifs IV and V, in Ad pol are essential for Ad DNA replication (34Roovers D.J. Overman P.F. Chen X.Q. Sussenbach J.S. Virology. 1991; 180: 273-284Crossref PubMed Scopus (16) Google Scholar, 35Roovers D.J. van der Lee F.M. van der Wees J. Sussenbach J.S. J. Virol. 1993; 67: 265-276Crossref PubMed Google Scholar, 36Chen M. Horwitz M.S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6116-6120Crossref PubMed Scopus (24) Google Scholar). Recently, a set of 22 alanine substitutions of conserved residues in the C-terminal part of Ad pol suggests an arrangement of conserved motifs in Ad pol similar to RB69 DNA polymerase (37Liu H. Naismith J.H. Hay R.T. J. Virol. 2000; 74: 11681-11689Crossref PubMed Scopus (24) Google Scholar). An additional motif, YXG(G/A), located N-terminal of motif II (12Delarue M. Poch O. Tordo N. Moras D. Argos P. Protein Eng. 1990; 3: 461-467Crossref PubMed Scopus (580) Google Scholar, 18Blanco L. Bernad A. Blasco M.A. Salas M. Gene (Amst.). 1991; 100: 27-38Crossref PubMed Scopus (192) Google Scholar) of the polymerase active site, was shown to be highly conserved among α-like DNA polymerases (38Truniger V. Lazaro J.M. Salas M. Blanco L. EMBO J. 1996; 15: 3430-3441Crossref PubMed Scopus (47) Google Scholar). Mutational analysis of this motif in φ29 DNA polymerase, which starts replication by protein-priming, led to the proposal that it is involved in the binding stability of the DNA template at the polymerization active site (38Truniger V. Lazaro J.M. Salas M. Blanco L. EMBO J. 1996; 15: 3430-3441Crossref PubMed Scopus (47) Google Scholar). Additionally, it was shown to be important for the formation of a stable complex between TP and DNA polymerase, resulting in transition defects from TP priming to DNA priming during replication of φ29 TP-DNA (39Truniger V. Blanco L. Salas M. J. Mol. Biol. 1999; 286: 57-69Crossref PubMed Scopus (27) Google Scholar). A multiple alignment of the YXG(G/A) motif in eukaryotic-type DNA-dependent DNA polymerases has been shown previously by Truniger et al. (38Truniger V. Lazaro J.M. Salas M. Blanco L. EMBO J. 1996; 15: 3430-3441Crossref PubMed Scopus (47) Google Scholar). In eukaryotic-type DNA polymerases, the motif has the consensus sequence YXG(G/A), but for the subclass of protein-primed DNA polymerases, the motif could be restricted to the consensus YXGG. For these DNA polymerases, including Ad pol, the highly conserved tyrosine residue is often an isoleucine. This led us to define the motif as (I/Y)XGG. Here, we report the detailed characterization of the (I/Y)XGG motif in Ad pol, which has been subjected to site-directed mutational analysis. We propose that the motif is involved in the stabilization of the template strand at the polymerase active site. During pTP-primed initiation, this indirectly affects the binding of the initiating nucleotide, as well as the transition of the initiation intermediate pTP-CAT from initiation to elongation, thereby leading to abortive replication. Based on the crystal structure of RB69, modeled with primer-template and dNTP, we propose a hydrophobic interaction between the conserved isoleucine and the ribose moiety of the nucleotide preceding the template base. All oligonucleotides, unlabeled nucleotides, [α-32P]dNTPs (3000 Ci/mmol), and [γ-32P]ATP (5000 Ci/mmol) were purchased from Amersham Pharmacia Biotech. T30 (5′-AATCCAAAATAAGGTATATTATTGATGATG) represents the first 30 nucleotides of the bottom strand of the adenovirus 5 genome, and T20 represents the first 20. D20 (5′-CATCATCAATAATATACCTT) is the complementary strand of T20. Labeling of D20 was performed with T4 polynucleotide kinase (Amersham Pharmacia Biotech) and [γ-32P]ATP. D20 was used for the 3′–5′-exonuclease assay on ssDNA. For the polymerase/exonuclease coupled assay, gel retardation, and 3′–5′-exonuclease assay on dsDNA, 5′-labeled D20 was hybridized to T30 or to T20. The hybrid molecules D20/T30 and T20/D20 were obtained by boiling oligonucleotides in 60 mmTris-HCl, pH 7.5, 200 mm NaCl, followed by slow cooling to room temperature. D20/T30 and D20/T20 were purified by 10% polyacrylamide-1× TBE gel electrophoresis. Ad 5 TP-DNA was isolated as described (40Coenjaerts F.E. van der Vliet P.C. Methods Enzymol. 1995; 262: 548-560Crossref PubMed Scopus (10) Google Scholar). A full-length Ad pol cDNA encoding amino acids 1–1199 (provided by Henk G. Stunnenberg (41Stunnenberg H.G. Lange H. Philipson L. van Miltenburg R.T. van der Vliet P.C. Nucleic Acids Res. 1988; 16: 2431-2444Crossref PubMed Scopus (61) Google Scholar)) was cloned in the EcoRI and SphI sites of the pFastBac donor plasmid. Site-directed mutagenesis was performed using the QuickChange method from Stratagene. The oligonucleotides for the polymerase chain reaction mutagenesis were as follows: for R661A, 5′-ATGCTGGCGGCCACGTAATCG and 5′-CGATTACGTGGCCGCCAGCAT; for I664S, 5′-TCTTCCACCGCGGGAGCTGGCG and 5′- CGCCAGCTCCCGCGGTGGAAGA; for I664Y, 5′-TCTTCCACCGCGGTAGCTGGCG and 5′-CGCCAGCTACCGCGGTGGAAGA; for G666A/G667A, 5′-GTAGCATCTTGCAGCGCGGATGCTC and 5′-GAGCATCCGCGCTGCAAGATGCTAC, with changes marked boldface type. The presence of the desired mutations was confirmed by complete sequencing of each mutant gene. The recombinant plasmids were transformed into DH10Bac competent cells, which contain the bacmid with a mini-attTn7 target site and a helper plasmid. The mini-Tn7 element on the pFastBac plasmid can transpose to the mini-attTn7 target site on the bacmid in the presence of transposition proteins provided by the helper plasmid. Colonies containing recombinant bacmids were identified by disruption of thelacZα gene. Bacmid DNA was isolated by means of a high molecular weight minipreparation. This DNA was then used to transfect insect cells with Lipofectin (Life Technologies) according to the manufacturer's manual. After 72 h of transfection, the recombinant baculoviruses were harvested and amplified for several rounds. Insect cells (Sf-9) were grown as monolayers on 167.5-cm2 plates in SF900 II medium (Life Technologies) at 27 °C. Plates were infected with recombinant baculovirus expressing the wild-type or mutant Ad pol when ∼80% confluence was reached. After 56 h of infection, cells were harvested and washed with ice-cold PBS. Cells were resuspended in a hypotonic lysis buffer containing 25 mm HEPES, pH 7.5, 10% glycerol, 5 mm KCl, 1 mm EDTA, 1 mmphenylmethylsulfonyl fluoride, 1 mm DTT, 2 µg/ml aprotinin, and 1 µg/ml leupeptin and placed on ice. After 10 min, cells were disrupted by 20 strokes of a Dounce homogenizer (B pestle), and NaCl was added to a final concentration of 200 mm. The lysate was cleared by ultracentrifugation at 25,000 rpm in a SW28 rotor for 30 min at 4 °C. For purification to near homogeneity, the lysate was loaded on a SP-Sepharose column, equilibrated with buffer A (25 mmHEPES, pH 7.5, 20% glycerol, 1 mm EDTA, 1 mmphenylmethylsulfonyl fluoride, 1 mm DTT) containing 200 mm NaCl. The column was washed extensively with buffer A, 200 mm NaCl and eluted with buffer A, 450 mm NaCl. Fractions were collected and analyzed on a 7.5% polyacrylamide, SDS gel followed by silver staining. Peak fractions were collected; dialyzed against buffer A, 100 mm NaCl; and loaded on a ssDNA-cellulose column (Sigma). After washing with buffer A, 150 mm NaCl, protein was eluted at 600 mmNaCl. Peak fractions were dialyzed against buffer B (25 mmHEPES, pH 8.0, 20% glycerol, 1 mm EDTA, 1 mmphenylmethylsulfonyl fluoride, 1 mm DTT) containing 100 mm NaCl and loaded onto a 1-ml Mono Q HR 5/5 column (Amersham Pharmacia Biotech). After washing with buffer B, 100 mm NaCl, protein was eluted in a gradient of buffer B, 100–500 mm NaCl. Peak fractions were collected, and the purity of the protein was estimated to be >95% by gel electrophoresis and Coomassie staining. pTP was a gift from Panagiotis N. Kanellopoulos. ΔN-DNA-binding protein was purified as described (42Dekker J. Kanellopoulos P.N. Loonstra A.K. van Oosterhout J.A. Leonard K. Tucker P.A. van der Vliet P.C. EMBO J. 1997; 16: 1455-1463Crossref PubMed Scopus (44) Google Scholar). The buffer used for dilution of the replication proteins contained 25 mm HEPES, pH 7.5, 20% glycerol, 120 mm NaCl, and 1 mg/ml bovine serum albumin (BSA). Partial duplex D20/T30 containing a stretch of 10 nucleotides protruding from the 5′-end was used as primer-template to study DNA-dependent DNA polymerization and 3′–5′-exonuclease activity. The reaction mixture (12.5 µl) contained 50 mm Tris-HCl, pH 7.5, 4% glycerol, 1 mm DTT, 1 mg/ml BSA, 1 mmMgCl2, 0.05 ng of 5′-labeled D20/T30, 12.5 ng of wild-type or mutant DNA polymerase, and the indicated amounts of dNTPs. Reactions were stopped after 10 min at 37 °C by the addition of sequencing loading buffer (10 mm EDTA, 98% formamide, and 0.025% bromphenol blue). Samples were analyzed on 8 m urea-20% polyacrylamide gel electrophoresis followed by autoradiography. Polymerization or 3′–5′- exonuclease activity were detected as an increase or decrease in size, respectively, of the 5′-labeled D20 primer. Exonucleolytic breakdown of ssDNA and dsDNA was tested using 5′-labeled D20 and 5′-labeled D20/T30, respectively. The reaction mixture (25 µl) contained 50 mm Tris-HCl, pH 7.5, 4% glycerol, 1 mm DTT, 1 mg/ml BSA, 50 mm NaCl, 1 mm MgCl2, 0.05 ng of ssDNA or dsDNA, and the reaction was started by the addition of 25 ng of wild-type polymerase or mutant polymerases. Incubation was at 37 °C, allowing conditions to be linear both in time and enzyme concentration. Reactions were stopped by the addition of sequencing loading buffer. After analysis by 8 m urea-20% polyacrylamide gel electrophoresis, the 3′–5′-exonuclease activity is measured as a decrease in size of the DNA by densitometry. From these data, the catalytic efficiency of the mutants (indicated in Table II) was calculated relative to wild-type Ad pol.Table IIProtein-primed activities of Ad wild-type and mutant polymerasesPolymeraseInitiation activity2-aData taken from initiation reactions on T30 and TP-DNA (the natural viral template) under standard conditions as described under “Experimental Procedures.” Numbers in parentheses indicate the percentage of activity in the presence of 10 µm unlabeled dCTP. Wild-type Ad pol initiation activity was set at 100%.pTP-pol2-bpTP-pol interaction, as determined by glycerol gradient centrifugation. +, wild-type (100 ± 20%) pTP-Ad pol interaction.interactionK m(app)2-cNumbers showK m(app) values of pTP deoxycytidylation with S.D. (n = 3 for each polymerase).TP-DNAssDNA%%µmdCTPWild type100100 (100)+8.3 ± 1.0I664S1614 (57)+22.6 ± 1.3I664Y2825 (63)+28.5 ± 0.8R661A2317 (54)+12.3 ± 2.7G666A/G667A711 (36)+76.5 ± 17.6The assays were performed as described under “Experimental Procedures.”2-a Data taken from initiation reactions on T30 and TP-DNA (the natural viral template) under standard conditions as described under “Experimental Procedures.” Numbers in parentheses indicate the percentage of activity in the presence of 10 µm unlabeled dCTP. Wild-type Ad pol initiation activity was set at 100%.2-b pTP-pol interaction, as determined by glycerol gradient centrifugation. +, wild-type (100 ± 20%) pTP-Ad pol interaction.2-c Numbers showK m(app) values of pTP deoxycytidylation with S.D. (n = 3 for each polymerase). Open table in a new tab The assays were performed as described under “Experimental Procedures.” Gel retardation was performed using 5′-labeled D20/T30 and 5′-labeled D20/T20. The binding reaction (20 µl) contained 25 mm HEPES, pH 7.5, 4% Ficoll, 1 mm EDTA, 55 mm NaCl, 4 mm DTT, 0.1 mg/ml BSA, 1 mm MgCl2, 0.05 ng of either 5′-labeled D20/T30 or 5′-labeled D20/T20, and the indicated amounts of Ad pol or the corresponding mutants. After incubation for 5 min at 4 °C, samples were loaded and separated on a 10% polyacrylamide-1× TBE gel at 4 °C. Gels were dried, autoradiographed, and quantified using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). Initiation of replication was performed in a standard incubation mixture of 25 µl in the presence of 25 mmHEPES, pH 7.5, 50 mm NaCl, 1.5 mmMgCl2, 1 mm DTT, 1 µg of BSA, 50 nm [α-32P]dCTP, and the indicated amounts of Ad pol and pTP. As template, either 0.6 µg of origin-containing T30 or 60 ng of TP-DNA were used. When TP-DNA was used, 250 ng of ΔN-DNA-binding protein was added per reaction. Initiation coupled to partial/truncated elongation was performed under similar conditions as initiation in the presence of the indicated concentrations of dCTP, dATP, and dTTP. No dGTP was added in the reaction mixture. Reactions were performed at 37 °C for 45 min and were stopped by adding EDTA to a final concentration of 80 mm. The samples were precipitated with 20% trichloroacetic acid on ice. Precipitates were washed with 5% trichloroacetic acid, dissolved in sample buffer, analyzed on an SDS-7.5% polyacrylamide gel, and autoradiographed. Replication products were quantified by densitometric analysis following exposure on a PhosphorImager. The K m(app) for pTP deoxynucleotidylation was determined by performing initiation assays with [α-32P]dCTP with wild-type and mutant polymerases using increasing concentrations of unlabeled dCTP (1–1000 µm). The K m(app) was calculated from three experiments. The standard incubation mixture (200 µl) for glycerol gradient analysis contained 2 µg of Ad pol, 1.2 µg of pTP, 25 mm HEPES, pH 7.5, 1 mm DTT, 1 mm MgCl2, and NaCl to a final concentration of 55 mm. After incubation for 30 min on ice, the mixture was layered on top of a 4.8-ml linear 10–30% (v/v) glycerol gradient containing 25 mm HEPES, pH 7.5, 1 mm DTT, 1 mm EDTA, 0.5 m NaCl, and 100 µg of BSA as an internal control. Gradients were centrifuged for 24 h at 50,000 rpm in a SW50 rotor at 4 °C. A control gradient with 1.2 µg of pTP was run under similar conditions. Fractions were collected from the bottom of the tube and analyzed on an SDS-7.5% polyacrylamide gel. BSA was visualized by silver staining, and pTP, Ad pol, and the pTP-pol complex were visualized by immunoblotting using an anti-pol and anti-pTP-pol antiserum (43Coenjaerts F.E. van Oosterhout J.A. van der Vliet P.C. EMBO J. 1994; 13: 5401-5409Crossref PubMed Scopus (45) Google Scholar) Quantitation of the relative amounts of pTP and pol present in each fraction was carried out by densitometry. In order to understand the role of the (I/Y)XGG motif in Ad pol, individual residues of this region were mutated (Fig.1) as described under “Experimental Procedures.” The isoleucine was changed into tyrosine (I664Y) as present in RB69, φ29, and most other cellular, bacterial, and viral DNA polymerases (39Truniger V. Blanco L. Salas M. J. Mol. Biol. 1999; 286: 57-69Crossref PubMed Scopus (27) Google Scholar) and also into serine (I664S) to study the effect of another nonconservative change. The two glycines are invariantly conserved among protein-primed DNA polymerases but the second glycine is often an alanine among bacterial, viral, and many cellular DNA polymerases (39Truniger V. Blanco L. Salas M. J. Mol. Biol. 1999; 286: 57-69Crossref PubMed Scopus (27) Google Scholar). Both glycines were changed into alanines, giving mutant polymerase G666A/G667A. A positive charge preceding the (I/Y)XGG motif (Arg661) appears to be specifically conserved among protein-primed and cellular DNA polymerases (38Truniger V. Lazaro J.M. Salas M. Blanco L. EMBO J. 1996; 15: 3430-3441Crossref PubMed Scopus (47) Google Scholar) and was changed into alanine (R661A). Construction of baculoviruses and expression and purification of the recombinant proteins was performed as described under “Experimental Procedures.” During purification, all mutant polymerases behaved essentially as wild-type Ad pol. A polymerase/exonuclease coupled assay was performed to study the coordination of both degradative and polymerization activities with the mutant polymerases. The functional coupling between synthesis and degradation on primer-template D20/T30 was assayed as a function of the dNTP concentration. As shown in Fig.2, 3′–5′-exonucleolytic digestion of the primer strand occurred in the absence of nucleotides. By the addition of increasing amounts of dNTPs, the equilibrium was shifted toward synthesis, exonucleolysis being competed by DNA polymerization. In the presence of 25 nm dNTPs, the wild-type Ad pol was able to extend D20/T30 until 27–28 nucleotides, and from 125 nmdNTPs full extension of the primer-template was accomplished (D30/T30). Whereas mutant polymerase R661A allowed full polymerization at approximately similar nucleotide concentrations as the wild-type enzyme (125 nm, Fig. 2), mutant polymerases I664S and I664Y required a 5-fold higher amount (Fig. 2 and TableI). On the other hand, mutant polymerase G666A/G667A required a 200-fold higher dNTP concentration compared with the wild-type enzyme for full polymerization (Fig. 2 and Table I). Furthermore, an increased exonuclease activity was observed for mutant polymerases I664S, I664Y, and G666A/G667A upon comparison of their degradation activities (Fig. 2, 0 nm lanes) with that of the wild-type polymerase, as can be seen by the higher intensity of the faster moving bands. The higher amount of dNTPs, required to fully elongate the primer-template for these three mutant polymerases, might be explained, at least partially, by their higher exonuclease activity. The 3′–5′-exonuclease activity was therefore determined on both ssDNA and dsDNA, and the results are quantified in Table I. Indeed, the exonuclease activity on primer-template DNA was increased for mutant polymerases I664S, I664Y, and G666A/G667A (TableI). Degradation of ssDNA by all mutant polymerases was slightly lower than that by the wild-type polymerase and proceeded in a distributive manner. An increased exonuclease activity on primer-template DNA might be the result of a lower DNA binding stability o
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