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• W2020162055 abstract "To better characterize the enzymatic activities required for human papillomavirus (HPV) DNA replication, the E1 helicases of HPV types 6 and 11 were produced using a baculovirus expression system. The purified wild type proteins and a version of HPV11 E1 lacking the N-terminal 71 amino acids, which was better expressed, were found to be hexameric over a wide range of concentrations and to have helicase and ATPase activities with relatively low values for Km(ATP) of 12 μm for HPV6 E1 and 6 μm for HPV11 E1. Interestingly, the value of Km(ATP) was increased 7-fold in the presence of the E2 transactivation domain. In turn, ATP was found to perturb the co-operative binding of E1 and E2 to DNA. Mutant and truncated versions of in vitro translated E1 were used to identify a minimal ATPase domain composed of the C-terminal 297 amino acids. This fragment was expressed, purified, and found to be fully active in ATP hydrolysis, single-stranded DNA binding, and unwinding assays, despite lacking the minimal origin-binding domain. To better characterize the enzymatic activities required for human papillomavirus (HPV) DNA replication, the E1 helicases of HPV types 6 and 11 were produced using a baculovirus expression system. The purified wild type proteins and a version of HPV11 E1 lacking the N-terminal 71 amino acids, which was better expressed, were found to be hexameric over a wide range of concentrations and to have helicase and ATPase activities with relatively low values for Km(ATP) of 12 μm for HPV6 E1 and 6 μm for HPV11 E1. Interestingly, the value of Km(ATP) was increased 7-fold in the presence of the E2 transactivation domain. In turn, ATP was found to perturb the co-operative binding of E1 and E2 to DNA. Mutant and truncated versions of in vitro translated E1 were used to identify a minimal ATPase domain composed of the C-terminal 297 amino acids. This fragment was expressed, purified, and found to be fully active in ATP hydrolysis, single-stranded DNA binding, and unwinding assays, despite lacking the minimal origin-binding domain. papillomavirus adenosine-5′-O-(3-thio)triphosphate bovine papillomavirus double-stranded DNA dithiothreitol glutathione S-transferase human papillomavirus 2-(N-morpholino)ethanesulfonic acid polyacrylamide gel electrophoresis polymerase chain reaction scintillation proximity assay single-stranded DNA E2 transactivation domain wild type nucleotide(s) The papillomaviruses (PVs)1 are small, nonenveloped DNA viruses that infect and replicate in the cutaneous or mucosal epithelia of humans and other mammals. There are over 100 types of human papillomavirus (HPV), which cause conditions ranging from plantar warts (HPV1) and genital warts (HPV6 and -11) to cervical cancer (HPV16, -18, and -31). HPV6 and -11 are also responsible for laryngeal papillomatosis, a rare but very serious infection of the respiratory tract (1Shah K.V. Howley P.M. Fields B.N. Knipe D.M. Howley P.M. Fields Virology. 3rd Ed. Lippincott-Raven Publishers, Philadelphia1996: 2077-2109Google Scholar). Antiviral agents capable of specifically inhibiting PV replication could play an important role in the treatment of these diseases, but none exist at this time. Despite their host and tissue specificity, all of the PV types share a common genomic organization. The closed, circular genome of ∼8000 base pairs codes for only 10 proteins: eight early proteins termed E1–E8 and two late proteins, L1 and L2, that make up the viral coat (2Howley P.M. Fields B.N. Knipe D.M. Howley P.M. Fields Virology. 3rd Ed. Lippincott-Raven Publishers, Philadelphia1996: 2045-2076Google Scholar). E1 and E2 are the only viral proteins required for HPV DNA replication (3Ustav M. Stenlund A. EMBO J. 1991; 10: 449-457Crossref PubMed Scopus (379) Google Scholar, 4Yang L. Li R. Mohr I.J. Clark R. Botchan M.R. Nature. 1991; 353: 628-632Crossref PubMed Scopus (240) Google Scholar). E2 is a sequence-specific DNA-binding protein that serves to regulate both transcription and DNA replication. E1, a DNA helicase, is the only PV protein that possesses enzymatic activity (5Yang L. Mohr I. Fouts E. Lim D.A. Nohaile M. Botchan M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5086-5090Crossref PubMed Scopus (221) Google Scholar) and is also the most highly conserved of the PV proteins. For these reasons, E1 has been considered as the most attractive molecular target for the development of antiviral agents (6Phelps W.C. Barnes J.A. Lobe D.C. Antiviral. Chem. Chemother. 1998; 9: 359-377Crossref PubMed Scopus (46) Google Scholar). During the initiation of PV DNA replication, E2 serves as a specificity factor to enhance binding of E1 monomers to the origin, which by themselves bind to double-stranded DNA with little sequence specificity (4Yang L. Li R. Mohr I.J. Clark R. Botchan M.R. Nature. 1991; 353: 628-632Crossref PubMed Scopus (240) Google Scholar, 7Frattini M.G. Laimins L.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12398-12402Crossref PubMed Scopus (114) Google Scholar, 8Lusky M. Hurwitz J. Seo Y.S. J. Biol. Chem. 1993; 268: 15795-15803Abstract Full Text PDF PubMed Google Scholar, 9Mohr I.J. Clark R. Sun S. Androphy E.J. MacPherson P. Botchan M.R. Science. 1990; 250: 1694-1699Crossref PubMed Scopus (327) Google Scholar, 10Sedman J. Stenlund A. EMBO J. 1995; 14: 6218-6228Crossref PubMed Scopus (160) Google Scholar, 11Sedman T. Sedman J. Stenlund A. J. Virol. 1997; 71: 2887-2896Crossref PubMed Google Scholar). E2 dimers bind with high specificity to DNA sequences within the origin and recruit to it E1 monomers, through direct protein-protein interactions (12Blitz I.L. Laimins L.A. J. Virol. 1991; 65: 649-656Crossref PubMed Google Scholar, 13Lusky M. Fontane E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6363-6367Crossref PubMed Scopus (73) Google Scholar). Upon binding to DNA, E1 monomers assemble into hexamers (14Liu J.S. Kuo S.R. Makhov A.M. Cyr D.M. Griffith J.D. Broker T.R. Chow L.T. J. Biol. Chem. 1998; 273: 30704-30712Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 15Fouts E.T., Yu, X. Egelman E.H. Botchan M.R. J. Biol. Chem. 1999; 274: 4447-4458Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), but the E1-E2 interaction is not maintained (16Lusky M. Hurwitz J. Seo Y.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8895-8899Crossref PubMed Scopus (79) Google Scholar, 17Sanders C.M. Stenlund A. EMBO J. 1998; 17: 7044-7055Crossref PubMed Scopus (93) Google Scholar). Thus, the role of E2 is to catalyze the assembly of hexameric E1 complexes specifically at the origin. ATP hydrolysis is also required for E1 hexamers to distort the origin and encircle single strands of DNA (18Gillette T.G. Lusky M. Borowiec J.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8846-8850Crossref PubMed Scopus (69) Google Scholar). Using in vitro translated HPV11 proteins, we have previously shown that ATP binding stimulates the E2-dependent association of E1 with the origin (19Titolo S. Pelletier A. Sauve F. Brault K. Wardrop E. White P.W. Amin A. Cordingley M.G. Archambault J. J. Virol. 1999; 73: 5282-5293Crossref PubMed Google Scholar). ATP binding does not affect the E2 binding step or the E1-E2 interaction but rather a subsequent process, either oligomerization and binding of E1 to DNA or the stability of the assembled E1-origin DNA complex (19Titolo S. Pelletier A. Sauve F. Brault K. Wardrop E. White P.W. Amin A. Cordingley M.G. Archambault J. J. Virol. 1999; 73: 5282-5293Crossref PubMed Google Scholar,20Titolo S. Pelletier A. Pulichino A.M. Brault K. Wardrop E. White P.W. Cordingley M.G. Archambault J. J. Virol. 2000; 74: 7349-7361Crossref PubMed Scopus (45) Google Scholar). ATP hydrolysis is not required in this case, since the nonhydrolyzable analog ATPγS and ADP have similar effects. For BPV, it has been demonstrated that ATP hydrolysis is required for displacement of E2 from the origin during assembly of E1 multimeric complexes (17Sanders C.M. Stenlund A. EMBO J. 1998; 17: 7044-7055Crossref PubMed Scopus (93) Google Scholar). Either concurrent with or after formation of E1 oligomers, the host polymerase α primase (21Park P. Copeland W. Yang L. Wang T. Botchan M.R. Mohr I.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8700-8704Crossref PubMed Scopus (127) Google Scholar, 22Bonne-Andrea C. Santucci S. Clertant P. Tillier F. J. Virol. 1995; 69: 2341-2350Crossref PubMed Google Scholar, 23Masterson P.J. Stanley M.A. Lewis A.P. Romanos M.A. J. Virol. 1998; 72: 7407-7419Crossref PubMed Google Scholar, 24Conger K.L. Liu J.S. Kuo S.R. Chow L.T. Wang T.S. J. Biol. Chem. 1999; 274: 2696-2705Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 25Amin A.A. Titolo S. Pelletier A. Fink D. Cordingley M.G. Archambault J. Virology. 2000; 272: 137-150Crossref PubMed Scopus (39) Google Scholar) and possibly also replication protein A (22Bonne-Andrea C. Santucci S. Clertant P. Tillier F. J. Virol. 1995; 69: 2341-2350Crossref PubMed Google Scholar, 26Han Y. Loo Y.M. Militello K.T. Melendy T. J. Virol. 1999; 73: 4899-4907Crossref PubMed Google Scholar) bind to E1, and a replication complex is formed together with additional host factors. We and others have identified subdomains of E1 as well as conserved amino acids that are required for a number of its DNA replication activities. We have recently shown that amino acids 166–649 of E1 efficiently support DNA replication in a cell-free system and thus must encode for all necessary binding and enzymatic activities requiredin vitro (25Amin A.A. Titolo S. Pelletier A. Fink D. Cordingley M.G. Archambault J. Virology. 2000; 272: 137-150Crossref PubMed Scopus (39) Google Scholar). In vivo, additional sequences spanning amino acids 82–128 (HPV11 E1) are required; these contain an extended nuclear localization sequence (27Lentz M.R. Pak D. Mohr I. Botchan M.R. J. Virol. 1993; 67: 1414-1423Crossref PubMed Google Scholar, 28Xiao X.L. Wilson V.G. J. Gen. Virol. 1994; 75: 2463-2467Crossref PubMed Scopus (19) Google Scholar) as well as a cyclin-binding motif and cyclin-dependent kinase phosphorylation sites (29McShan G.D. Wilson V.G. J. Gen. Virol. 1997; 78: 171-177Crossref PubMed Scopus (19) Google Scholar, 30Cueille N. Nougarede R. Mechali F. Philippe M. Bonne-Andrea C. J. Virol. 1998; 72: 7255-7262Crossref PubMed Google Scholar, 31Ma T. Zou N. Lin B.Y. Chow L.T. Harper J.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 382-387Crossref PubMed Scopus (114) Google Scholar). A minimal DNA binding/origin recognition sequence was mapped to approximately amino acids 140–300 for BPV E1 (32Thorner L.K. Lim D.A. Botchan M.R. J. Virol. 1993; 67: 6000-6014Crossref PubMed Google Scholar, 33Sarafi T.R. McBride A.A. Virology. 1995; 211: 385-396Crossref PubMed Scopus (60) Google Scholar, 34Leng X. Ludes-Meyers J.H. Wilson V.G. J. Virol. 1997; 71: 848-852Crossref PubMed Google Scholar, 35Chen G. Stenlund A. J. Virol. 1998; 72: 2567-2576Crossref PubMed Google Scholar) (amino acids 185–345 for HPV11 E1), although the entire C terminus of the protein (amino acids 186–649) is required to observe stable binding of HPV11 E1 to the origin (20Titolo S. Pelletier A. Pulichino A.M. Brault K. Wardrop E. White P.W. Cordingley M.G. Archambault J. J. Virol. 2000; 74: 7349-7361Crossref PubMed Scopus (45) Google Scholar, 36Sun Y. Han H. McCance D.J. J. Gen. Virol. 1998; 79: 1651-1658Crossref PubMed Scopus (24) Google Scholar). An HPV11 E1 truncation consisting of the C-terminal amino acids 353–649 retains the ability to bind to E2 or the p70 subunit of polymerase α primase and to oligomerize into hexamers (19Titolo S. Pelletier A. Sauve F. Brault K. Wardrop E. White P.W. Amin A. Cordingley M.G. Archambault J. J. Virol. 1999; 73: 5282-5293Crossref PubMed Google Scholar, 20Titolo S. Pelletier A. Pulichino A.M. Brault K. Wardrop E. White P.W. Cordingley M.G. Archambault J. J. Virol. 2000; 74: 7349-7361Crossref PubMed Scopus (45) Google Scholar, 25Amin A.A. Titolo S. Pelletier A. Fink D. Cordingley M.G. Archambault J. Virology. 2000; 272: 137-150Crossref PubMed Scopus (39) Google Scholar). Within this C-terminal fragment of E1 are four conserved regions termed A, B, C, and D (see Fig. 1), which are highly similar to the functionally related T antigens of SV40 and polyomaviruses (37Clertant P. Seif I. Nature. 1984; 311: 276-279Crossref PubMed Scopus (88) Google Scholar). The conserved region A lies in a minimal E1 oligomerization sequence located within amino acids 353–416 (see Fig. 1). Three motifs characteristic of superfamily 3 of NTP-binding proteins are located between amino acids 478 and 525 of HPV11 E1 (see Fig. 1) (38Gorbalenya A.E. Koonin E.V. Wolf Y.I. FEBS Lett. 1990; 262: 145-148Crossref PubMed Scopus (295) Google Scholar). Two of these motifs (motifs a and b) correspond to the Walker A and B boxes involved in binding the substrate nucleotide triphosphate-magnesium complex (39Walker J.E. Saraste M. Runswick M.J. Gay N.J. EMBO J. 1982; 1: 945-951Crossref PubMed Scopus (4234) Google Scholar), while the exact function of the third motif (motif c) remains unclear. Mutations of conserved residues in all three motifs have been shown to significantly decrease the ATPase activity of the superfamily 3 helicase Rep68, encoded by the adeno-associated virus (40Walker S.L. Wonderling R.S. Owens R.A. J. Virol. 1997; 71: 6996-7004Crossref PubMed Google Scholar). Mutation of a highly conserved lysine in motif a (Lys484 in HPV11 E1) has been shown to abrogate the ATPase activity of BPV E1 (41MacPherson P. Thorner L. Parker L.M. Botchan M. Virology. 1994; 204: 403-408Crossref PubMed Scopus (35) Google Scholar) and HPV11 E1 (42Rocque W.J. Porter D.J.T. Barnes J.A. Dixon E.P. Lobe D.C. Su J.L. Willard D.H. Gaillard R. Condreay J.P. Clay W.C. Hoffman C.R. Overton L.K. Pahel G. Kost T.A. Phelps W.C. Protein Expression Purif. 2000; 18: 148-159Crossref PubMed Scopus (22) Google Scholar), as also observed for many other ATPases (43Schulz G.E. Curr. Opin. Struct. Biol. 1992; 2: 61-67Crossref Scopus (299) Google Scholar). Most studies on the enzymatic activities of purified E1 have been carried out using the BPV enzyme, purified from baculovirus-infected insect cells (5Yang L. Mohr I. Fouts E. Lim D.A. Nohaile M. Botchan M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5086-5090Crossref PubMed Scopus (221) Google Scholar, 41MacPherson P. Thorner L. Parker L.M. Botchan M. Virology. 1994; 204: 403-408Crossref PubMed Scopus (35) Google Scholar, 44Seo Y.S. Muller F. Lusky M. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 702-706Crossref PubMed Scopus (176) Google Scholar, 45Santucci S. Bonne-Andrea C. Clertant P. J. Gen. Virol. 1995; 76: 1129-1140Crossref PubMed Scopus (18) Google Scholar) or Escherichia coli (15Fouts E.T., Yu, X. Egelman E.H. Botchan M.R. J. Biol. Chem. 1999; 274: 4447-4458Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 46Sedman J. Stenlund A. J. Virol. 1998; 72: 6893-6897Crossref PubMed Google Scholar). Although the HPV E1 proteins are 40–50% identical in primary sequence to BPV E1 and presumably have very similar structures and mechanisms of action, it has proven very difficult to isolate sufficient quantities of purified HPV enzymes to characterize their enzymatic activities. Very low levels of expression, compounded by proteolysis and contamination with other proteins, have commonly been observed. Of the few reports on the isolation of HPV E1 (42Rocque W.J. Porter D.J.T. Barnes J.A. Dixon E.P. Lobe D.C. Su J.L. Willard D.H. Gaillard R. Condreay J.P. Clay W.C. Hoffman C.R. Overton L.K. Pahel G. Kost T.A. Phelps W.C. Protein Expression Purif. 2000; 18: 148-159Crossref PubMed Scopus (22) Google Scholar, 47Hughes F.J. Romanos M.A. Nucleic Acids Res. 1993; 21: 5817-5823Crossref PubMed Scopus (109) Google Scholar, 48Raj K. Stanley M.A. J. Gen. Virol. 1995; 76: 2949-2956Crossref PubMed Scopus (12) Google Scholar, 49Jenkins O. Earnshaw D. Sarginson G. Del Vecchio A. Tsai J. Kallender H. Amegadzie B. Browne M. J. Gen. Virol. 1996; 77: 1805-1809Crossref PubMed Scopus (25) Google Scholar), in only one case was sufficient material obtained for a basic characterization of helicase activity and biophysical properties (42Rocque W.J. Porter D.J.T. Barnes J.A. Dixon E.P. Lobe D.C. Su J.L. Willard D.H. Gaillard R. Condreay J.P. Clay W.C. Hoffman C.R. Overton L.K. Pahel G. Kost T.A. Phelps W.C. Protein Expression Purif. 2000; 18: 148-159Crossref PubMed Scopus (22) Google Scholar). Kuo et al. (50Kuo S.R. Liu J.S. Broker T.R. Chow L.T. J. Biol. Chem. 1994; 269: 24058-24065Abstract Full Text PDF PubMed Google Scholar) have reported the isolation of epitope-tagged HPV11 E1 that is functional for in vitro DNA replication assays, but the enzymatic activities of their E1 preparations have not been described. In this report, we present our characterization of the enzymatic activities of HPV11 and -6 E1 helicases. We have made extensive use of an N-terminal truncated form of E1, composed of amino acids 72–649, which we have shown previously is as active as wild type E1 in supporting HPV DNA replication in vitro. We report here that this truncated E1 is expressed in significantly greater yield than full-length E1 and has improved biophysical properties. We show that, in contrast to previous reports (41MacPherson P. Thorner L. Parker L.M. Botchan M. Virology. 1994; 204: 403-408Crossref PubMed Scopus (35) Google Scholar, 42Rocque W.J. Porter D.J.T. Barnes J.A. Dixon E.P. Lobe D.C. Su J.L. Willard D.H. Gaillard R. Condreay J.P. Clay W.C. Hoffman C.R. Overton L.K. Pahel G. Kost T.A. Phelps W.C. Protein Expression Purif. 2000; 18: 148-159Crossref PubMed Scopus (22) Google Scholar, 45Santucci S. Bonne-Andrea C. Clertant P. J. Gen. Virol. 1995; 76: 1129-1140Crossref PubMed Scopus (18) Google Scholar, 49Jenkins O. Earnshaw D. Sarginson G. Del Vecchio A. Tsai J. Kallender H. Amegadzie B. Browne M. J. Gen. Virol. 1996; 77: 1805-1809Crossref PubMed Scopus (25) Google Scholar), our purified E1 preparations have a relatively low value of Km(ATP), well below the physiological concentration of ATP. We show that high concentrations of the E2 transactivation domain raise the apparent value of Km(ATP) and also demonstrate that the binding of ATP-Mg to E1 weakens its interaction with E2. The perturbation of E1-E2 binding by physiological concentrations of ATP suggests a mechanism by which some of the energy gained by tight binding of ATP is used to destabilize the E1-E2 complex, which forms transiently during the initiation of viral DNA replication. We also show that E1-(353–649), which encodes motifs for enzymatic activity but lacks the previously identified minimal origin-DNA binding region, is fully active in ATPase, helicase, and ssDNA binding assays. Thus, while the N-terminal half of E1 is necessary for replication from the HPV origin, all sequences necessary for proper folding, oligomerization, and enzymatic catalysis are within the C terminus. The complete E1 open reading frame from HPV11 was amplified from plasmid pCR3-E1 (19Titolo S. Pelletier A. Sauve F. Brault K. Wardrop E. White P.W. Amin A. Cordingley M.G. Archambault J. J. Virol. 1999; 73: 5282-5293Crossref PubMed Google Scholar). The forward primer was 5′-CGC GGA TCC AGG ATG CAT CAC CAT CAC CAT CAC GCG GAC GAT TCA CGT ACA GAA AAT GAG-3′, and the reverse was GG CTG AAT TCA TAA AGT TCT AAC AAC T-3′, inserting 6 histidines immediately after the initial methionine. Similarly, E1-(72–649) and E1-(353–649) were amplified from plasmid pTM1-E1 (19Titolo S. Pelletier A. Sauve F. Brault K. Wardrop E. White P.W. Amin A. Cordingley M.G. Archambault J. J. Virol. 1999; 73: 5282-5293Crossref PubMed Google Scholar) with the same reverse primer but with the following forward primers, respectively: 5′-CGC GGA TCC AGG ATG CAT CAC CAT CAC CAT CAC GCG GAT GCT CAT TAT GCG ACT GTG CAG GAC-3′ and 5′-CGC GGA TCC AGG ATG CAT CAC CAT CAC CAT CAC GAC AGT CAA TTT AAA TTA ACT GAA ATG GTG C-3′. To express the corresponding catalytically inactive mutant E1 proteins, similar PCR amplifications were performed on the appropriate mutant E1 template (19Titolo S. Pelletier A. Sauve F. Brault K. Wardrop E. White P.W. Amin A. Cordingley M.G. Archambault J. J. Virol. 1999; 73: 5282-5293Crossref PubMed Google Scholar). The PCR products were then cut with BamHI and EcoRI, subcloned between theBamHI and EcoRI sites of pFASTBAC1 (Life Technologies, Inc.), and transposed into the baculovirus genome according to the manufacturer's instructions. E1-containing recombinant baculovirus genomes were transfected into SF9 insect cells, and baculovirus-containing supernatants were amplified for large scale protein production. The gene for HPV6 E1 was amplified by PCR from a clinical condyloma sample and subcloned into pCR3.1. PCR primers used were 5′-CAA CGA TGG CGG ACG ATT CAG GTA CAG-3′ (forward, nt 827–853) and 5′-TGC TTC GGA CAC CT-3′ (reverse, nt 2923–2910). The baculovirus expression construct was made by the same procedure as for HPV11 E1, using the same forward and reverse primers for PCR. The DNA sequence matched that reported for HPV6a E1 (GenBankTM accession number L41216) except for eight silent mutations (nt 1297 C to A, nt 1495 T to A, nt 1540 C to T, nt 2170 C to T, nt 2363 T to C, nt 2396 T to C, nt 2530 G to A, and nt 2548 T to C) and two mutations encoding conservative changes (nt 1670 G to T, changing Val280 to Leu, and nt 1741 C to A, changing Asp303 to Glu). 2L. Bourgon and L. Doyon, personal communication. The amino acid sequence for HPV6a E1 is 88% identical to that for HPV11 E1. SF21 cells were maintained in SFM-900 medium (Life Technologies) at cell densities between 1 and 2 × 106/ml. Infections were carried out for 48–72 h using a multiplicity of infection of 5. Cells were harvested and frozen rapidly in liquid nitrogen before being stored at −80 °C. All subsequent steps were performed at 0–4 °C. For nuclear extraction, frozen cell pellets were thawed and resuspended in one volume of cell lysis buffer (20 mm Tris, pH 8, 5 mm β-mercaptoethanol, 5 mm KCl, 1 mm MgCl2, 1 mm Pefabloc (Pentapharm Ltd.), 1 μg/ml pepstatin, 1 μg/ml leupeptin, and 1 μg/ml antipain) for 15 min and then broken with a Dounce homogenizer (20 strokes for 40 ml, pestle B) followed by centrifugation at 2500 × g for 20 min. Supernatant (cytosol) was discarded, and nuclei were resuspended to 1.4 volumes with extraction buffer A (20 mm Tris, pH 8, 5 mm β-mercaptoethanol, 2 mm Pefabloc, 2 μg/ml pepstatin, 2 μg/ml leupeptin, and 2 μg/ml antipain), followed by the addition of an equal volume of extraction buffer B (20 mm Tris, pH 8, 5 mm β-mercaptoethanol, and 0.02% Triton X-100 for full-length E1 or the same buffer supplemented with NaCl to a final concentration of 450 mm for truncated proteins). The nuclei were incubated with rocking for 30 min before ultracentrifugation at 148,000 × g for 45 min. Glycerol was added to the supernatant to a final concentration of 10%, and the extract was frozen rapidly on dry ice and stored at −80 °C. All steps were performed at 4 °C. A 5-ml Hi-Trap chelating column was charged with NiSO4according to the manufacturer's instructions (Amersham Pharmacia Biotech) and then washed with equilibration buffer (20 mmTris, pH 8, 5 mm β-mercaptoethanol, 500 mmNaCl, 10 mm imidazole, and 10% glycerol). Nuclear extracts from 5 liters of culture were thawed rapidly, the NaCl concentration was adjusted to 500 mm for full-length E1 that was initially extracted in low salt, and the material was loaded onto the Hi-Trap column. The column was washed with 50 ml of equilibration buffer followed by 10 volumes of washing buffer (equilibration buffer with 50 mm imidazole). His-tagged E1 was eluted in 1-ml fractions with equilibration buffer containing 180 mmimidazole and dialyzed in 20 mm MES, pH 7.0, 500 mm NaCl, 1 mm DTT, 0.05 mm EDTA, and 10% glycerol before being frozen on dry ice and stored at −80 °C. E1 truncations were eluted with 250 mm(E1-(72–649)) or 350 mm (E1-(353–649)) imidazole and were not dialyzed prior to freezing, since dilution of the protein into assay buffers was found to give insignificant residual imidazole concentrations. E1 preparations were tested for the presence of DNA using the fluorescent dye PicoGreen (Molecular Probes, Inc., Eugene, OR), according to published procedures (51Singer V.L. Jones L.J. Yue S.T. Haugland R.P. Anal. Biochem. 1997; 249: 228-238Crossref PubMed Scopus (641) Google Scholar). The concentrations of purified proteins were determined using the Coomassie Plus reagent (Pierce), with bovine serum albumin serving as a standard. Bismaleimidohexane cross-linking of purified HPV11 E1-(72–649) and subsequent analysis on Weber-Osborn polyacrylamide gels (52Weber K. Osborn M. J. Biol. Chem. 1969; 244: 4406-4412Abstract Full Text PDF PubMed Google Scholar) were performed as described previously forin vitro translated protein (20Titolo S. Pelletier A. Pulichino A.M. Brault K. Wardrop E. White P.W. Cordingley M.G. Archambault J. J. Virol. 2000; 74: 7349-7361Crossref PubMed Scopus (45) Google Scholar). A velocity run for HPV11 E1-(72–649) was carried out using the XL-A analytical ultracentrifuge (Beckman Coulter), in an An-60 Ti rotor at 40,000 rpm and 11 °C. E1-(72–649) was exchanged into a buffer composed of 20 mmTris, 500 mm NaCl, 10% glycerol, 1 mm DTT, and 0.1 mm EDTA, pH 8.0, using a NAP-5 column (Amersham Pharmacia Biotech). The protein was diluted to ∼40 μg/ml (600 nm) and analyzed using a 1.2-cm double sector charcoal-filled Epon centerpiece and quartz windows pretreated with Sigmacoat silconizing agent (Sigma). Data were acquired at 230 nm and at 0.005-cm intervals, with five replicate readings taken at each point. Scans were spaced as closely as the absorbance optics would allow, approximately every 2 min. E1-(72–649) was found to be stable over the time required to acquire velocity data, although it began to lose activity after several hours at 4–11 °C. Buffer density was determined to be 1.05659 ± 0.00007 g/ml at 22 °C using a pyknometer and corrected to a value of 1.05851 g/ml at 11 °C (53Laue T.M. Shah B.D. Ridgeway T.M. Pelletier S.L. Harding S. Rowe A. Analytical Ultracentrifugation in Biochemistry and Polymer Science. Redwood Press Ltd., Melksham, UK1992: 90-125Google Scholar). The partial specific volume for E1-(72–649) (0.7285 cm3/g) and the solvent viscosity (1.810 centipoise) at 11 °C were calculated using the program Sednterp (53Laue T.M. Shah B.D. Ridgeway T.M. Pelletier S.L. Harding S. Rowe A. Analytical Ultracentrifugation in Biochemistry and Polymer Science. Redwood Press Ltd., Melksham, UK1992: 90-125Google Scholar). Data were analyzed with the programs DCDT+ 1.13 (54Stafford III., W.F. Anal. Biochem. 1992; 203: 295-301Crossref PubMed Scopus (518) Google Scholar, 55Philo J.S. Anal. Biochem. 2000; 279: 151-163Crossref PubMed Scopus (237) Google Scholar) and SVEDBERG 6.38 (56Philo J.S. Biophys. J. 1997; 72: 435-444Abstract Full Text PDF PubMed Scopus (212) Google Scholar), which gave similar results. E2-dependent binding of E1 to the HPV11 origin was measured using a modification of the procedure previously used for in vitro translated proteins (19Titolo S. Pelletier A. Sauve F. Brault K. Wardrop E. White P.W. Amin A. Cordingley M.G. Archambault J. J. Virol. 1999; 73: 5282-5293Crossref PubMed Google Scholar). Binding reactions (80 μl) contained ∼0.4 ng of probe DNA, 2 μg of salmon sperm DNA, and the indicated amounts of E1 and E2. Radiolabeled probe DNA was obtained by PCR amplification of the HPV11 origin from plasmid pN9. HPV11 E2 was expressed in SF21 insect cells infected with baculovirus Ac11E2 (obtained from R. Rose, University of Rochester) and purified by DNA affinity chromatography in a procedure similar to that of Seo et al. (57Seo Y.S. Muller F. Lusky M. Gibbs E. Kim H.Y. Phillips B. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2865-2869Crossref PubMed Scopus (152) Google Scholar). After 1-h binding reactions, detection was performed by a modification of the previously described procedure in which protein A scintillation proximity assay beads (SPA beads) were used in place of protein A-Sepharose. Anti-E1-coated SPA beads were added to binding reactions, and after an additional 1 h, plates were spun briefly to pellet beads and counted using a TopCount NXT microplate scintillation counter (Packard Instruments). Blanks were subtracted before calculating relative activities. M13mp18 ssDNA was obtained by infection of E. coli according to standard procedures (58Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989)Molecular Cloning: A Laboratory Manual, 2nd Ed., pp. 4.21–32, 11.23–26, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar). Substrate oligonucleotides were synthesized (trityl-off) on an Applied Biosystems 394 DNA/RNA synthesizer and purified by gel electrophoresis (58Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989)Molecular Cloning: A Laboratory Manual, 2nd Ed., pp. 4.21–32, 11.23–26, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar). To synthesize the substrate used for most of the work in this report, 125 pmol of the purified oligonucleotide 5′-TTC CCA GTC ACG TTG T-3′ was mixed with an equimolar amount of M13mp18 in 500 μl of TEN buffer (10 mm Tris, 80 mm NaCl, 1 mm EDTA), heated in a water bath to 95 °C, and then allowed to cool slowly overnight. dCTP was then added (to give 230 μm), together with [α-33P]dATP (125 pmol or 250 μCi) and unlabeled dATP (400 pmol), to give a final volume of 700 μl, and the oligonucleotide sequence was extended with the Klenow fragment at 30 °C to give the final sequence 5′-TTC CCA GTC ACG ACG TTG TAA AAC-3′ (radiolabeled residues in boldface). The labeled partial duplex was purified using a freshly packed 50-ml column of Sepharose 4B equilibrated in TEN buffer. Pooled partial duplex fractions (∼10 ml) were concentrated to ∼2 ml using Centriprep 50 filters (Millipore Corp.). The typical y" @default.
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• W2020162055 date "2001-06-01" @default.
• W2020162055 modified "2023-09-27" @default.
• W2020162055 title "Characterization of Recombinant HPV6 and 11 E1 Helicases" @default.
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• W2020162055 doi "https://doi.org/10.1074/jbc.m101932200" @default.
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