Characterization of an African Swine Fever Virus 20-kDa DNA Polymerase Involved in DNA Repair
1997; Elsevier BV; Volume: 272; Issue: 49 Linguagem: Inglês
10.1074/jbc.272.49.30899
ISSN1083-351X
AutoresMariano Oliveros, Rafael J. Yáñez‐Muñoz, María Salas, José Salas, Eladio Viñuela, Luis Blanco,
Tópico(s)Viral gastroenteritis research and epidemiology
ResumoAfrican swine fever virus (ASFV) encodes a novel DNA polymerase, constituted of only 174 amino acids, belonging to the polymerase (pol) X family of DNA polymerases. Biochemical analyses of the purified enzyme indicate that ASFV pol X is a monomeric DNA-directed DNA polymerase, highly distributive, lacking a proofreading 3′-5′-exonuclease, and with a poor discrimination against dideoxynucleotides. A multiple alignment of family X DNA polymerases, together with the extrapolation to the crystal structure of mammalian DNA polymerase β (pol β), showed the conservation in ASFV pol X of the most critical residues involved in DNA binding, nucleotide binding, and catalysis of the polymerization reaction. Therefore, the 20-kDa ASFV pol X most likely represents the minimal functional version of an evolutionarily conserved pol β-type DNA polymerase core, constituted by only the "palm" and "thumb" subdomains. It is worth noting that such an "unfingered" DNA polymerase is able to handle templated DNA polymerization with a considerable high fidelity at the base discrimination level. Base excision repair is considered to be a cellular defense mechanism repairing modified bases in DNA. Interestingly, the fact that ASFV pol X is able to conduct filling of a single nucleotide gap points to a putative role in base excision repair during the ASFV life cycle. African swine fever virus (ASFV) encodes a novel DNA polymerase, constituted of only 174 amino acids, belonging to the polymerase (pol) X family of DNA polymerases. Biochemical analyses of the purified enzyme indicate that ASFV pol X is a monomeric DNA-directed DNA polymerase, highly distributive, lacking a proofreading 3′-5′-exonuclease, and with a poor discrimination against dideoxynucleotides. A multiple alignment of family X DNA polymerases, together with the extrapolation to the crystal structure of mammalian DNA polymerase β (pol β), showed the conservation in ASFV pol X of the most critical residues involved in DNA binding, nucleotide binding, and catalysis of the polymerization reaction. Therefore, the 20-kDa ASFV pol X most likely represents the minimal functional version of an evolutionarily conserved pol β-type DNA polymerase core, constituted by only the "palm" and "thumb" subdomains. It is worth noting that such an "unfingered" DNA polymerase is able to handle templated DNA polymerization with a considerable high fidelity at the base discrimination level. Base excision repair is considered to be a cellular defense mechanism repairing modified bases in DNA. Interestingly, the fact that ASFV pol X is able to conduct filling of a single nucleotide gap points to a putative role in base excision repair during the ASFV life cycle. Despite the variety of existing DNA polymerases, there are a few basic principles that are common to all these enzymes, irrespective of their role either in DNA replication or in DNA repair. The basic chemistry of each individual reaction always involves a pair of divalent metal ions that are coordinated by carboxylate residues. Such a two-metal ion mechanism, originally proposed by Beese and Steitz (1Beese L.S. Steitz T.A. EMBO J. 1991; 10: 25-33Crossref PubMed Scopus (943) Google Scholar) and probably extrapolative to all nucleotidyltransferases (2Steitz T.A. Curr. Opin. Struct. Biol. 1993; 3: 31-38Crossref Scopus (206) Google Scholar, 3Holm L. Sander C. Trends Biochem. Sci. 1995; 20: 345-347Abstract Full Text PDF PubMed Scopus (241) Google Scholar, 4Martin G. Keller W. EMBO J. 1996; 15: 2593-2603Crossref PubMed Scopus (168) Google Scholar), appears to be either evolutionarily conserved or acquired by convergent evolution of nonhomologous proteins. In addition to the general deoxynucleotidyl transfer mechanism, it appears that some structural convergency could apply also for the interaction with DNA, a common substrate; an overall view of the crystal structures available for DNA-dependent polymerases always shows a hand-shaped structure, with "thumb," "palm," and "fingers" subdomains, defining at least one cleft for holding DNA (5Ollis D.L. Brick R. Hamlin R. Xuong N.G. Steitz T.A. Nature. 1985; 313: 762-766Crossref PubMed Scopus (784) Google Scholar, 6Beese L.S. Derbyshire V. Steitz T.A. Science. 1993; 260: 352-355Crossref PubMed Scopus (462) Google Scholar, 7Sousa R. Chung Y.J. Rose J.P. Wang B.C. Nature. 1993; 364: 593-599Crossref PubMed Scopus (349) Google Scholar, 8Jacobo-Molina A. Ding J. Nanni R. Clark Jr., A.D. Lu X. Tantillo C. Williams R.L. Kamer G. Ferris A.L. Clark P. Hizi A. Hughes S.H. Arnold E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6320-6324Crossref PubMed Scopus (1129) Google Scholar, 9Pelletier H. Sawaya M.R. Kumar A. Wilson S.H. Kraut J. Science. 1994; 264: 1891-1903Crossref PubMed Scopus (768) Google Scholar, 10Kim Y. Eom S.H. Wang J. Lee D.-S. Suh S.W. Steitz T.A. Nature. 1995; 376: 612-616Crossref PubMed Scopus (334) Google Scholar, 11Georgiadis M.M. Jessen S.M. Ogata C.M. Telesnitsky A. Goff S.P. Hendrickson W.A. Structure. 1995; 3: 879-892Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 12Kiefer J.R. Mao C. Hansen C.J. Rasehore S.L. Hogrefe H.H. Braman J.C. Beese L.S. Structure. 1997; 5: 95-108Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Moreover, both DNA replicases and DNA repair enzymes are often multienzymatic proteins, having built-in nucleolytic activities that exist as individual structural modules, separated from the polymerization domain. Thus, the paradigmatic Escherichia coli DNA polymerase I (pol I) 1The abbreviations used are: pol, polymerase; ASFV, African swine fever virus; BER, base excision repair; TdT, terminal transferase; IPTG, isopropyl-1-thio-β-d-galactopyranoside; NTA, nitrilotriacetic acid; DTT, dithiothreitol; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; HhH, helix-hairpin-helix; AP, apurinic/apyrimidinic; kb, kilobase pair(s); bp, base pair(s); dd, dideoxy. has a proofreading 3′-5′-exonuclease, and a 5′-3′-exonuclease to remove the RNA from the Okazaki fragments (13Kornberg A. Baker T.A. DNA Replication. 2nd Ed. Freeman, San Francisco1992Google Scholar). Most DNA replicases are also endowed with a proofreading 3′-5′-exonuclease domain, having an evolutionarily conserved pol I-type active site (14Bernad A. Blanco L. Lázaro J.M. Martin G. Salas M. Cell. 1989; 59: 219-228Abstract Full Text PDF PubMed Scopus (374) Google Scholar). Reverse transcriptases have an RNase H activity, required for second strand DNA synthesis (reviewed in Telesnitsky and Goff (15Telesnitsky A. Goff S.P. Skalka A.M. Goff S.P. Reverse Transcriptases. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993: 49-84Google Scholar)). Even pol β, the smallest of the known DNA polymerases (39 kDa), has a 8-kDa N-terminal domain that can excise a 5′-terminal base-free deoxyribose phosphate residue from incised abasic sites by a β-elimination mechanism (16Matsumoto Y. Kim K. Science. 1995; 269: 699-702Crossref PubMed Scopus (656) Google Scholar, 17Piersen C.E. Prasad R. Wilson S.H. Lloyd R.S. J. Biol. Chem. 1996; 271: 17811-17815Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). However, considering that DNA synthesis has to be productively coordinated with these various degradative functions, specific differences in the geometry of the enzyme/DNA interaction should be expected. Additional differences in complexity and/or specificity at the structural level among DNA polymerases could be the consequence of the necessity to interact with accessory proteins to increase processivity, or with specialized molecules (as tRNA or terminal proteins) acting as primers. On the other hand, the maximal simplicity at the structural/functional level could be envisioned for a DNA polymerase involved in a minimal DNA synthesis reaction, exemplified by the repair of a single nucleotide gap. Such a "custom made" DNA nucleotidyltransferase would require three carboxylate residues involved in metal binding and catalysis, a DNA binding site providing a very precise interaction with the templating base and the primer terminus, necessary for an efficient and faithful catalysis, and several residues devoted to bind and select the appropriate dNTP substrates. Such a minimal DNA-synthesizing enzyme should be able to dissociate promptly after catalysis, to facilitate the subsequent action of a ligase. In this sense, additional DNA binding subdomains or motifs other than those forming the catalytic core would be inconvenient. African swine fever virus (ASFV), an enveloped deoxyvirus (170 kb), which causes a fatal disease to domestic pigs (18Viñuela E. Becker Y. African Swine Fever. Martinus Nijhoff, Boston1987: 31-49Google Scholar, 19Wilkinson P.J. Pensaert M.B. Virus Infections of Porcines. Elsevier Sciences Publishers B.V., Amsterdam, Holland1989: 17-37Google Scholar, 20Costa J. Darai G. Molecular Biology of Iridoviruses. Kluwer Academic, Dordrecht, Holland1990: 247-270Crossref Google Scholar), not only encodes a eukaryotic-type (family B) DNA polymerase involved in viral DNA replication (21Moreno M.A. Carrascosa A.L. Ortı́n J. Viñuela E. J. Gen. Virol. 1978; 93: 253-258Crossref Scopus (30) Google Scholar, 22Rodrı́guez J.M. Yáñez R.J. Rodrı́guez J.F. Viñuela E. Salas M.L. Gene (Amst.). 1993; 136: 103-110Crossref PubMed Scopus (27) Google Scholar, 23Martins A. Ribeiro G. Marques M.I. Costa J.V. Nucleic Acids Res. 1994; 22: 208-213Crossref PubMed Scopus (20) Google Scholar) but, as shown in this study, also codes for a new member of the pol X family of DNA polymerases, a peculiar group that includes pol β and terminal transferase (TdT) from vertebrates (24Ito J. Braithwaite D.K. Nucleic Acids Res. 1991; 19: 4045-4057Crossref PubMed Scopus (297) Google Scholar), mitochondrial pol β from protozoans (25Torri A.F. Englund P.T. J. Biol. Chem. 1995; 270: 3495-3497Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), and DNA polymerase IV (pol IV) from yeast (26Bork P. Ouzounis C. Sander C. Scharf M. Schneider R. Sonnhammer E. Protein Sci. 1992; 1: 1677-1690Crossref PubMed Scopus (99) Google Scholar, 27Prasad R. Widen S.G. Singhal R.K. Watkins J. Prakash L. Wilson S.H. Nucleic Acids Res. 1993; 21: 5301-5307Crossref PubMed Scopus (74) Google Scholar). Interestingly, the ASFV pol X is only 20 kDa but efficiently catalyzes a single nucleotide gap repair reaction. A three-dimensional structure prediction for ASFV pol X based upon extrapolation to the crystal structure of pol β (9Pelletier H. Sawaya M.R. Kumar A. Wilson S.H. Kraut J. Science. 1994; 264: 1891-1903Crossref PubMed Scopus (768) Google Scholar, 28Sawaya M.R. Pelletier H. Kumar A. Wilson S.H. Kraut J. Science. 1994; 264: 1930-1935Crossref PubMed Scopus (403) Google Scholar, 29Davies II, J.F. Almassy R.J. Hostomska Z. Ferre R.A. Hostomsky Z. Cell. 1994; 76: 1123-1133Abstract Full Text PDF PubMed Scopus (175) Google Scholar) is discussed, as well as a putative role in BER of the viral DNA. Unlabeled nucleotides were purchased from Pharmacia P-L Biochemicals. [γ-32P]ATP (3000 Ci/mmol) and [α-32P]dATP (3000 Ci/mmol) were obtained from Amersham International Plc. Restriction endonucleases, T4 polynucleotide kinase, T4 DNA ligase, and Klenow fragment were from Boehringer Mannheim. Oligonucleotides RO-17 (5′-GCGCGCGGATCCATGTTAACGCTTATTCAAGGAAAAA) and RCP-2 (5′-CGCGCGCTGCAGTTATAAACGTTTCTTAGGTATGCGA) were used for polymerase chain reaction-amplification of ASFV gene O174L. Oligonucleotides SP1 (5′-GATCACAGTGAGTAC) and SP1p (5′-GATCACAGTGAGTAG), differing in the 3′-terminal base, were used as primer strands. Oligonucleotide SP1c+6 (5′-TCTATTGTACTCACTGTGATC-3′), that has a 5′-terminal extension of six nucleotides in addition to the sequence complementary to SP1, oligonucleotides SP1c+6(A) (5′-TCTATAGTACTCACTGTGATC), SP1c+6(G) (5′-TCTATGGTACTCACTGTGATC) and SP1c+6(C) (5′-TCTATCGTACTCACTGTGATC), differing in the first template base (indicated in their respective names) respect to the sequence of SP1c+6, oligonucleotide SP1c+18(g1) (5′-ACTGGCCGTCGTTTTACTGTACTCACTGTGATC) that has a 5′-terminal extension of 18 nucleotides in addition to the sequence complementary to SP1, and oligonucleotide SP1c+18(g6) (5′-ACTGGCCGTCGTTCTATTGTACTCACTGTGATC), that has a 5′-terminal extension of 12 nucleotides in addition to the sequence of SP1c+6, were used as template strands. Oligonucleotide D(g1) (5′-GTAAAACGACGGCCAGT), complementary to the last 17 5′-nucleotides of SP1c+18(g1), oligonucleotide D(g6) (5′-ACGACGGCCAGT), complementary to the last 12 5′-nucleotides of SP1c+18(g6), and oligonucleotides D(g1)P and D(g6)P, 5′-phosphorylated versions of oligonucleotides D(g1) and D(g6), respectively, were used as downstream oligonucleotides to construct gapped structures of 1 or 6 nucleotides. All the oligonucleotides described above were obtained from Life Technologies, Inc. Oligonucleotides SP1 and SP1p were 5′-labeled with [γ-32P]ATP and T4 polynucleotide kinase and purified by electrophoresis on 8 m urea, 20% polyacrylamide gels. To analyze the DNA-dependent polymerization activity of the protein on different substrates (template-primer and gapped structures), the labeled oligonucleotides (primer) were hybridized to one (template) or two different (template and downstream) oligonucleotides in the presence of 0.2 m NaCl and 60 mm Tris-HCl (pH 7.5). Multiple alignment of the DNA polymerase sequences shown in Fig. 1 was done in two steps. The initial alignment of the putative ASFV pol X protein with sequences of TdT and pol β from different sources was done using the computer programs PILEUP and PRETTY from the University of Wisconsin Genetics Computer Group (30Deveraux J. Haeberli P. Smithies O. Nucleic Acids Res. 1984; 12: 387-395Crossref PubMed Scopus (12363) Google Scholar). At the second step, the multiple alignment obtained was adjusted manually, refining it on the basis of the secondary structure elements of rat pol β, as deduced from its crystal structure (9Pelletier H. Sawaya M.R. Kumar A. Wilson S.H. Kraut J. Science. 1994; 264: 1891-1903Crossref PubMed Scopus (768) Google Scholar, 28Sawaya M.R. Pelletier H. Kumar A. Wilson S.H. Kraut J. Science. 1994; 264: 1930-1935Crossref PubMed Scopus (403) Google Scholar). The open reading frame containing the putative DNA polymerase X geneO174L from ASFV (31Yáñez R.J. Rodrı́guez J.M. Nogal M.L. Yuste L. Enrı́quez C. Rodrı́guez J.F. Viñuela E. Virology. 1995; 208: 249-278Crossref PubMed Scopus (360) Google Scholar) was cloned into the pRSET-A bacterial expression vector, which allows the expression of recombinant proteins as fusions with a multifunctional leader peptide containing a hexahistidyl sequence for purification on Ni+2-affinity resins (32Kroll D.J. Abedel-Malek Abdel-Hafiz H. Marcell T. Simpson S. Chen C.Y. Gutierrez-Hartmann A. Lustbader J.W. Hoeffler J.P. DNA Cell Biol. 1993; 12: 441-453Crossref PubMed Scopus (77) Google Scholar). First, open reading frame O174L was polymerase chain reaction-amplified from a plasmid clone of ASFV strain BA71V (31Yáñez R.J. Rodrı́guez J.M. Nogal M.L. Yuste L. Enrı́quez C. Rodrı́guez J.F. Viñuela E. Virology. 1995; 208: 249-278Crossref PubMed Scopus (360) Google Scholar) using oligonucleotides RO-17 (with a 5′ extension that includes aBamHI restriction site) and RCP-2 (with a 5′ extension that includes a PstI restriction site). The resulting 0.5-kb polymerase chain reaction product was cloned at theBamHI/PstI sites of vector pRSET-A. The E. coli strain JM109 was used as a host for transformation. The construction of the recombinant expression plasmid, named pRSET-pol X was confirmed by DNA sequencing. Expression of the His-tagged pO174L protein was carried out in the E. coli strain BL21(DE3) pLysS, which contains the T7 RNA polymerase gene under the control of the isopropyl β-d-thiogalactopyranoside (IPTG)-inducible lacUV5 promoter, and a plasmid constitutively expressing T7 lysozyme (33Studier F.W. Moffat B.A. J. Mol. Biol. 1986; 189: 113-130Crossref PubMed Scopus (5151) Google Scholar, 34Studier F.W. J. Mol. Biol. 1991; 219: 37-44Crossref PubMed Scopus (701) Google Scholar). Cells were transformed with plasmid pRSET-O174L and grown overnight in LB medium at 37 °C. Flasks containing LB broth were inoculated with 0.1 volume of an overnight culture of E. coli and incubated in a rotatory shaker at 35 °C until the absorbance at 595 nm reached 0.7. Then, IPTG (Sigma) was added to a final concentration of 0.4 mm and incubation was continued for 3 h at 35 °C. Cells were collected by centrifugation for 15 min at 1,900 × g, and washed twice with buffer A (50 mm phosphate buffer, pH 7.5, 500 mm NaCl, 20 mm imidazole). After resuspension in the same buffer, cells were sonicated on ice. Then, the suspension was cleared by centrifugation for 10 min at 1,900 × g, at 4 °C, and an aliquot of the supernatant was withdrawn as a "total extract" fraction. The recombinant 20.3-kDa protein was soluble under these conditions, since it remained in the supernatant after a new centrifugation for 20 min at 14,500 × g, at 4 °C. Induction, overproduction, and solubility of the recombinant protein was analyzed by polyacrylamide gel electrophoresis (PAGE) in the presence of SDS, in a 7–20% polyacrylamide gradient, and visualized by Coomassie Blue staining. Ni-NTA agarose beads (QIAgen), previously equilibrated in buffer A, were added to the soluble fraction containing the recombinant protein, obtained as described above. After stirring for 2 h at 4 °C, the resin was loaded into a 1.6-cm diameter column, and extensively washed with buffer A. The recombinant ASFV protein pO174L was eluted from the column with buffer B (50 mm phosphate buffer, pH 7.5, 500 mm NaCl, 500 mm imidazole). The eluate was loaded onto a 5-ml glycerol gradient (15–30%) containing 50 mm Tris-HCl, pH 7.5, 20 mm ammonium sulfate, 180 mm NaCl, 1 mm EDTA, and 7 mm 2-β-mercaptoethanol, and centrifuged at 62,000 rpm (Beckman SW.50 rotor) for 26 h, at 4 °C. After centrifugation, 20 fractions were collected from the bottom of the tube, examined in Coomassie Blue-stained gels, and tested for DNA polymerase activity on activated DNA. The assay was carried out essentially as described previously (35Karawya E. Swack J.A. Wilson S. Anal. Biochem. 1983; 135: 318-325Crossref PubMed Scopus (56) Google Scholar). The protein samples were electrophoresed in 7–20% gradient SDS-PAGE gels containing 1.5 mg/ml activated calf thymus DNA (Pharmacia Biotech Inc.) as template-primer, followed by in situ renaturation of proteins and incubation of the gel in a DNA polymerase assay mixture. Prior to renaturation, the gel was washed twice with 50 mmTris-HCl, pH 7.5, for 15 min at 4 °C. Renaturation was allowed to occur during 3 h at 4 °C in buffer C (50 mmTris-HCl, pH 7.5, 6 mm (AcO)2Mg, 40 mm KCl, 16% glycerol, 0.01 mm EDTA, 1 mm dithiothreitol (DTT), and 400 μg/ml bovine serum albumin (BSA)). In situ polymerization was assayed with buffer D (buffer C plus 2 μm each dNTP and 1.2 nm [α-32P]dATP) for 12 h at 30 °C. After washing unincorporated [α-32P]dATP from the gel, and in situ precipitation of the DNA with buffer E (5% trichloroacetic acid, 1% sodium pyrophosphate), the gel was dried and the activity bands (radioactively labeled) were detected by autoradiography. The incubation mixture contained, in 25 μl, 50 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 1 mm DTT, 4% glycerol, 0.1 mg/ml BSA, 100 nm [α-32P]dATP and 100 nm of the other three dNTPs, 1.25 μg of activated calf thymus DNA as a substrate, and different fractions (2 μl) from a glycerol gradient containing ASFV pol X DNA polymerase. When indicated, the activated DNA was omitted, and the activating MgCl2 was either omitted or substituted by MnCl2. Butylanilino-dATP, butylphenyl-dGTP, phosphonoacetic acid, and aphidicolin (specific inhibitors of eukaryotic-type (family B) DNA polymerases), or ddNTPs (specific inhibitors of family X DNA polymerases) were added at the indicated concentrations. After incubation for 5 min at 37 °C, the reaction was stopped by adding 10 mm EDTA, 0.1% SDS, and the samples were filtered through Sephadex G-50 spin columns in the presence of 0.1% SDS. The excluded volume, corresponding to the labeled DNA, was counted (Cerenkov radiation). Polymerization activity was calculated as the amount of incorporated dNMP. The reaction mixture contained, in 10 μl, 50 mm Tris-HCl, pH 7.5, 10 mmMgCl2, 1 mm DTT, 4% glycerol, 0.1 mg/ml BSA, 200 ng of purified ASFV pol X, and 5′-labeled SP1 (3.2 nm) as single-stranded substrate. After incubation for different times at 37 °C, the reactions were stopped by adding EDTA up to 10 mm. Samples were analyzed by 8 m urea, 20% PAGE and autoradiography. 3′-5′-Exonuclease activity would be detected as a decrease in the size (15 mer) of the 5′-labeled SP1 primer. Terminal transferase activity was evaluated by using 5′-labeled SP1 as substrate. The incubation mixture contained, in 10 μl, 50 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 1 mm DTT, 4% glycerol, 0.1 mg/ml BSA, different concentrations (up to 400 μm) of the four dNTPs, 200 ng of purified ASFV pol X, and 3.2 nm of 5′-labeled SP1. After incubation for different times at 37 °C, the reactions were stopped by adding EDTA up to 10 mm. Samples were analyzed by 8m urea, 20% PAGE and autoradiography. DNA-dependent polymerization was assayed on different primer-template structures, obtained by hybridization of 5′-labeled SP1 (or SP1p) to the template oligonucleotide indicated in each case. The incubation mixture contained, in 10 μl, 50 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 1 mm DTT, 4% glycerol, 0.1 mg/ml BSA, 3.2 nm of the hybrid indicated in each case, and the indicated amount and concentration of ASFV pol X DNA polymerase and the four dNTPs, respectively. After incubation for different times at 37 °C, the reactions were stopped by adding EDTA up to 10 mm. Samples were analyzed by 8 m urea, 20% PAGE and autoradiography. Quantitation was done by densitometric analysis of the band(s) corresponding to primer extension products. The catalytic efficiency and nucleotide substrate specificity of ASFV pol X was assayed on the template-primer SP1/SP1c+6 (3.2 nm), essentially as described above. In this case, insertion of the nucleotide complementary to the two first template bases (providing various concentrations of either dATP (up to 10 μm) or ddATP (up to 10 μm) or ATP (up to 400 μm) as the sole nucleotide) was assayed in the presence of 200 ng of ASFV pol X and either MgCl2 (10 mm) or MnCl2 (4 mm) as metal activator. To analyze the base specificity (nucleotide insertion fidelity) of ASFV pol X, the oligonucleotide SP1 was hybridized to four variants of the SP1c+6 template oligonucleotide (SP1c+6 (T), SP1c+6 (G), SP1c+6 (C), SP1c+6 (A), differing in the first template base. Nucleotide insertion on each hybrid structure (3.2 nm) was comparatively studied as described above, by providing 200 ng of ASFV pol X and various concentrations of either the correct dNTP (up to 20 μm) or each of the three wrong dNTPs (up to 400 μm). To obtain "gapped" structures of defined length, a third oligonucleotide was hybridized to the corresponding template-primer oligonucleotide (3.2 nm). Thus, a 1-nucleotide gapped structure was obtained by hybridization of SP1 and D(g1)P oligonucleotides to the template oligonucleotide SP1c+18(g1); 6-nucleotide gapped structures were obtained by hybridization of SP1 and either D(g6) or D(g6)P to the template oligonucleotide SP1c+18(g6). DNA polymerization on these structures was essentially as described above, in the presence of the indicated amounts of ASFV pol X and different sets of dNTPs. When indicated, T4 DNA ligase (0.5 units) was added to the reaction mixture to seal the repaired DNA gap. 5′-Labeled oligo SP1 or SP1p either alone or hybridized to different oligonucleotides were used to analyze the interaction of ASFV pol X with DNA. The incubation mixture, in a final volume of 20 μl, contained 12 mm Tris-HCl, pH 7.5, 1 mm EDTA, 20 mm ammonium sulfate, 0.1 mg/ml BSA, 20 ng of ASFV pol X, and 3.2 nm of different single or hybrid oligonucleotides. After incubation for 5 min at 4 °C, the samples were subjected to electrophoresis in 4% (w/v) polyacrylamide gels (80:1 monomer:bis), containing 12 mmTris acetate, pH 7.5, and 1 mm EDTA, and run at 4 °C in the same buffer at 8 V/cm, essentially as described previously (36Carthew R.W. Chodosch L.A. Sharp P.A. Cell. 1985; 43: 439-448Abstract Full Text PDF PubMed Scopus (478) Google Scholar). After autoradiography, DNA polymerase-DNA complexes were detected as a mobility shift (retardation) in the migrating position of the labeled DNA. By complete DNA sequencing of the avirulent ASFV BA71V strain and subsequent data base searches for amino acid sequence similarities, it had been reported that the ASFV open reading frame designated as O174L potentially encoded a new member of the pol X family of DNA polymerases (31Yáñez R.J. Rodrı́guez J.M. Nogal M.L. Yuste L. Enrı́quez C. Rodrı́guez J.F. Viñuela E. Virology. 1995; 208: 249-278Crossref PubMed Scopus (360) Google Scholar). This prediction was tested by the multiple alignment of this sequence with those of pol β and TdT from several sources (24Ito J. Braithwaite D.K. Nucleic Acids Res. 1991; 19: 4045-4057Crossref PubMed Scopus (297) Google Scholar, 25Torri A.F. Englund P.T. J. Biol. Chem. 1995; 270: 3495-3497Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) and yeast pol IV (37Oliver S.G. van der Aart Q.J. Agosttini-Carbone M.L. Aigle M. Alberghina L. Alexandraki D. Antoine G. Anwar R. Ballesta J.P. Benit P. et al.Nature. 1992; 357: 38-46Crossref PubMed Scopus (715) Google Scholar), as representative members of DNA polymerase family X. A critical aspect to obtain the multiple alignment shown in Fig. 1 was the final adjustment carried out, as described under "Materials and Methods," on the basis of the reported crystal structure of rat pol β (9Pelletier H. Sawaya M.R. Kumar A. Wilson S.H. Kraut J. Science. 1994; 264: 1891-1903Crossref PubMed Scopus (768) Google Scholar, 28Sawaya M.R. Pelletier H. Kumar A. Wilson S.H. Kraut J. Science. 1994; 264: 1930-1935Crossref PubMed Scopus (403) Google Scholar, 29Davies II, J.F. Almassy R.J. Hostomska Z. Ferre R.A. Hostomsky Z. Cell. 1994; 76: 1123-1133Abstract Full Text PDF PubMed Scopus (175) Google Scholar). Thus, in general, the regions having a higher sequence similarity correspond to three-dimensional portions having a defined secondary structure, whereas the most variable regions among the DNA polymerases aligned correspond to random-coiled portions connecting well ordered regions. It has been reported that the N-terminal ∼170 residues of yeast pol IV bear no homology to mammalian pol β (26Bork P. Ouzounis C. Sander C. Scharf M. Schneider R. Sonnhammer E. Protein Sci. 1992; 1: 1677-1690Crossref PubMed Scopus (99) Google Scholar). According to the alignment shown here, the N-terminal 170 amino acid residues of yeast pol IV can be significantly aligned with the N-terminal portion of TdTs (similarity is indicated with gray boxes in Fig. 1). This finding together with the fact that this extra N-terminal domain had already been reported to be absent in pol β in previous alignments of the pol X family (24Ito J. Braithwaite D.K. Nucleic Acids Res. 1991; 19: 4045-4057Crossref PubMed Scopus (297) Google Scholar, 38Matsukage A. Nishikawa K. Ooi T. Seto Y. Yamaguchi M. J. Biol. Chem. 1987; 262: 8960-8962Abstract Full Text PDF PubMed Google Scholar) (see also Fig. 1), points to the existence of some specific structural/functional relationships among yeast pol IV and TdTs. Like pol β, ASFV pol X lacks this N-terminal extension, but also its two neighbor subdomains, i.e. the so-called "8 kDa" and "fingers" (green and yellow areas, respectively, in Fig.1), present in all the other sequences aligned. The alignment of ASFV pol X starts at the C-terminal half of α-helix I, at the very end of the "fingers" subdomain, and extends through the "palm" and "thumb" subdomains (red and magenta areas, respectively, in Fig. 1). The regions corresponding to some stretches of secondary structure elements of both palm and thumb subdomains (as J-1-K-2, 5-M-N-6–7, and O-310; see Fig. 1 for nomenclature) could be aligned without significant gaps among the different sequences, including ASFV pol X. This conservation at the primary structure level is probably the reflect of superior order constraints to form a conserved catalytic core. Another stretch of secondary structure elements (L-3–4) involves the same number of amino acid residues among ASFV pol X and pol βs, whereas in the case of TdTs and yeast pol IV, several amino acids (from 5 to 28 residues) are inserted among these elements. Mitochondrial pol β differs from cellular pol βs in larger intervening sequences connecting secondary structure elements, particularly that (26 amino acid residues) between β-strand 7 and α-helix O. Contrarily, and contributing to its reduced size, ASFV pol X lacks some intervening amino acid sequences, such as that among β-strand 2 and α-helix L, which is particularly extensive in the case of yeast pol IV. In support of its functional significance, the inclusion of ASFV pol X in this multiple alignment outlines a very limited number (15Telesnitsky A. Goff S.P. Skalka A.M. Goff S.P. Reverse Transcriptases. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993: 49-84Google Scholar) of invariant residues (indicated inwhite letters in Fig. 1), that include the catalytic triad of aspartates involved in metal binding (reviewed in Joyce and Steitz (39Joyce C.M. Steitz T.A. Annu. Rev. Biochem. 1994; 63: 777-822Crossref PubMed Scopus (579) Google Scholar)). Interestingly, the thumb is the subdomain showing the highest similarity among the sequences compared, containing eight invariant residues. On the contrary, the palm subdomain, that contains seven in
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