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• W2007346089 abstract "We present a preliminary biochemical characterization of two simian virus 40 mutants that affect different T antigen replication functions. SV40 T antigen mutants dl1135 (Δ17-27 amino acids) and 5080 (P-L) have been studied extensively with regard to their ability to transform cells in culture and induce tumors in transgenic mice. Both mutants are defective for viral DNA replication in vivo. In order to assess in more detail the molecular basis for the in vivo replication defects of 5080 and dl1135, we expressed the mutant proteins using the baculovirus system and purified them by immunoaffinity chromatography. With each of the purified proteins, we examined some of the biochemical activities of T antigen required for replication, viz. ATPase, binding to the origin of replication (ori) and assembly on ori, DNA helicase and unwinding, and replication in in vitro assays. Consistent with previous studies, we found that the 5080 protein is defective for multiple biochemical activities including ATPase, helicase, ori-specific unwinding, and ATP-induced hexamerization. However, this mutant retains some sequence-specific DNA binding activity. In contrast, the dl1135 protein exhibited significant levels of activity in all assays, including the ability to drive SV40 DNA replication in vitro. Thus, dl1135 is one of several mutants with an altered amino-terminal domain which can replicate DNA in vitro, but not in vivo. Thus, while the 5080 mutation affects a T antigen enzymatic function directly required for viral DNA synthesis, dl1135 may alter an activity required to prepare the cell for viral replication. We present a preliminary biochemical characterization of two simian virus 40 mutants that affect different T antigen replication functions. SV40 T antigen mutants dl1135 (Δ17-27 amino acids) and 5080 (P-L) have been studied extensively with regard to their ability to transform cells in culture and induce tumors in transgenic mice. Both mutants are defective for viral DNA replication in vivo. In order to assess in more detail the molecular basis for the in vivo replication defects of 5080 and dl1135, we expressed the mutant proteins using the baculovirus system and purified them by immunoaffinity chromatography. With each of the purified proteins, we examined some of the biochemical activities of T antigen required for replication, viz. ATPase, binding to the origin of replication (ori) and assembly on ori, DNA helicase and unwinding, and replication in in vitro assays. Consistent with previous studies, we found that the 5080 protein is defective for multiple biochemical activities including ATPase, helicase, ori-specific unwinding, and ATP-induced hexamerization. However, this mutant retains some sequence-specific DNA binding activity. In contrast, the dl1135 protein exhibited significant levels of activity in all assays, including the ability to drive SV40 DNA replication in vitro. Thus, dl1135 is one of several mutants with an altered amino-terminal domain which can replicate DNA in vitro, but not in vivo. Thus, while the 5080 mutation affects a T antigen enzymatic function directly required for viral DNA synthesis, dl1135 may alter an activity required to prepare the cell for viral replication. Simian virus 40 (SV40) has been a useful model for the study of DNA replication and transcription as well as for better understanding the mechanisms of tumorigenesis. The 708-amino-acid large tumor (T) antigen is the major regulator of productive and transforming infections(1DePamphilis M.C. Bradley M.K. Salzman N.P. The Papovaviridae. Vol. 1. Plenum Publishing Corp., New York1986: 99-246Crossref Google Scholar, 2Fanning E. J. Virol. 1992; 66: 1289-1293Crossref PubMed Google Scholar, 3Fanning E. Doefler W. Bohm P. Malignant Transformation by DNA Viruses Molecular Mechanisms. VCH Verlagsgesellschaft mbH, Weinheim1992: 1-19Google Scholar, 4Cosman D.J. Tevethia M.J. Virology. 1981; 112: 605-624Crossref PubMed Scopus (26) Google Scholar, 5Spence S.L. Pipas J.M. J. Virol. 1994; 68: 4227-4240Crossref PubMed Google Scholar, 6Spence S.L. Pipas J.M. Virology. 1994; 204: 200-209Crossref PubMed Scopus (34) Google Scholar, 7Fanning E. Knippers R. Annu. Rev. Biochem. 1992; 61: 55-85Crossref PubMed Scopus (458) Google Scholar). Many of the biochemical functions of T antigen have been mapped to specific domains of the polypeptide (Fig. 1)(1DePamphilis M.C. Bradley M.K. Salzman N.P. The Papovaviridae. Vol. 1. Plenum Publishing Corp., New York1986: 99-246Crossref Google Scholar, 8Prives C. Cell. 1990; 61: 735-738Abstract Full Text PDF PubMed Scopus (109) Google Scholar). These include: DNA binding (residues 131-259), ATPase/DNA helicase activity(302-627), and DNA polymerase α-primase binding (1-82; 260-517)(3Fanning E. Doefler W. Bohm P. Malignant Transformation by DNA Viruses Molecular Mechanisms. VCH Verlagsgesellschaft mbH, Weinheim1992: 1-19Google Scholar, 9Pipas J.M. J. Virol. 1992; 66: 3979-3985Crossref PubMed Google Scholar). The first step in viral DNA synthesis is the sequence-specific binding of T antigen within the origin of replication. The region of the origin of replication contains three T antigen binding sites (site I, site II, and site III) in decreasing order of affinity(1DePamphilis M.C. Bradley M.K. Salzman N.P. The Papovaviridae. Vol. 1. Plenum Publishing Corp., New York1986: 99-246Crossref Google Scholar, 10Borowiec J.A. Dean F.B. Bullock P.A. Hurwitz J. Cell. 1990; 60: 181-184Abstract Full Text PDF PubMed Scopus (287) Google Scholar). Site II, the core ori, is part of a 64-base pair sequence that is necessary and sufficient for viral DNA replication; site I and site III can stimulate viral replication(10Borowiec J.A. Dean F.B. Bullock P.A. Hurwitz J. Cell. 1990; 60: 181-184Abstract Full Text PDF PubMed Scopus (287) Google Scholar, 11Guo Z.-S. Gutierrez C. Heine U. Sogo J. DePamphilis M. Mol. Cell. Biol. 1989; 9: 3593-3602Crossref PubMed Scopus (44) Google Scholar). Both site I and site II have pentameric 5′-GAGGC-3′ sequences, but in different arrangements. Additionally, site II has two other regions, a 15-base pair inverted repeat on the early side and a 17-base pair A/T-rich region on the late side, which are involved in ori melting and untwisting of the DNA helix, respectively(12Parsons R. Anderson M.E. Tegtmeyer P. J. Virol. 1990; 64: 509-518Crossref PubMed Google Scholar, 13Borowiec J.A. Hurwitz J. EMBO J. 1988; 7: 3149-3158Crossref PubMed Scopus (167) Google Scholar, 14Deb S. DeLucia A.L. Koff A. Tsui S. Tegtmeyer P. Mol. Cell. Biol. 1986; 6: 4578-4584Crossref PubMed Scopus (92) Google Scholar). A monomer of T antigen can bind to one pentameric sequence in the absence of ATP, but ATP not only increases T antigen binding severalfold but it also induces a conformational change in T antigen such that 12 monomers of T antigen become organized into a two-lobed structure spanning the entire core ori(10Borowiec J.A. Dean F.B. Bullock P.A. Hurwitz J. Cell. 1990; 60: 181-184Abstract Full Text PDF PubMed Scopus (287) Google Scholar, 15Dean F.B. Dodson M. Echols H. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8981-8985Crossref PubMed Scopus (77) Google Scholar, 16Deb S.P. Tegtmeyer P. J. Virol. 1987; 61: 3649-3654Crossref PubMed Google Scholar, 17Borowiec J.A. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 64-68Crossref PubMed Scopus (78) Google Scholar, 18Mastrangelo I.A. Hough P.V.C. Wall J.S. Dodson M. Dean F.B. Hurwitz J. Nature. 1989; 338: 658-662Crossref PubMed Scopus (263) Google Scholar). The T antigen dodecamer complex then melts the origin. The ability of T antigen to bind to and melt site II is regulated by phosphorylation (19-21). An intact zinc finger region, which maps between amino acid residues 302 and 320, is required for hexamer assembly on the origin and for melting of the origin DNA(22Loeber G. Stenger J.E. Ray S. Parsons R.E. Anderson M.E. Tegtmeyer P. J. Virol. 1991; 65: 3167-3174Crossref PubMed Google Scholar). The presynthesis complex consists of T antigen assembled as a double hexamer in the presence of ATP onto a site II duplex subsequently bound by replication protein A (RP-A), a mammalian single-stranded DNA binding protein(7Fanning E. Knippers R. Annu. Rev. Biochem. 1992; 61: 55-85Crossref PubMed Scopus (458) Google Scholar, 16Deb S.P. Tegtmeyer P. J. Virol. 1987; 61: 3649-3654Crossref PubMed Google Scholar, 17Borowiec J.A. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 64-68Crossref PubMed Scopus (78) Google Scholar, 23Dodson M. Dean F.B. Bullock Echols H. Hurwitz J. Science. 1987; 238: 964-967Crossref PubMed Scopus (98) Google Scholar). With the association of DNA polymerase α-primase, hexameric T antigen in complex with RPA unwinds the DNA bidirectionally in a reaction driven by the hydrolysis of ATP(7Fanning E. Knippers R. Annu. Rev. Biochem. 1992; 61: 55-85Crossref PubMed Scopus (458) Google Scholar, 24Gannon J.V. Lane D.P. New Biol. 1990; 2: 84-92PubMed Google Scholar, 25Dornreiter I. Erdile L.F. Gilbert I.U. von Winkler D. Kelly T.J. Fanning E. EMBO J. 1992; 11: 769-776Crossref PubMed Scopus (285) Google Scholar, 26Wessel R. Schweizer J. Stahl H. J. Virol. 1992; 66: 804-815Crossref PubMed Google Scholar, 27Collins K.L. Kelly T.J. Mol. Cell. Biol. 1991; 11: 2108-2115Crossref PubMed Google Scholar). SV40 mutants dl1135 and 5080 have been studied extensively with regard to their ability to transform cells in culture and induce tumors in transgenic mice(28Peden K.W.C. Srinivasan A. Farber J.M. Pipas J.M. Virology. 1989; 168: 13-21Crossref PubMed Scopus (59) Google Scholar, 29Pipas J.M. Peden K.W.C. Nathans D. Mol. Cell. Biol. 1983; 3: 203-213Crossref PubMed Scopus (118) Google Scholar, 30Srinivasan A. Peden K.W.C. Pipas J.M. J. Virol. 1989; 63: 5459-5463Crossref PubMed Google Scholar, 31Symonds H.S. McCarthy S.A. Chen J. Pipas J.M. van Dyke T. Mol. Cell. Biol. 1993; 13: 3255-3265Crossref PubMed Scopus (55) Google Scholar, 32Van Dyke T.A. Cancer Biol. 1994; 5: 47-60PubMed Google Scholar). The mutant 5080 carries an amino acid substitution of P584L located within a stretch of hydrophobic residues near the carboxyl-terminal boundary of the ATPase/p53 binding domain. Previous studies indicated that the 5080 T antigen expressed in transformed murine cell lines was defective for ATPase activity and showed an aberrant pattern of oligomerization and phosphorylation(33Tack L.C. Cartwright C.A. Wright J.H. Eckhart W. Peden K.W.C. Srinivasan A. Pipas J.M. J. Virol. 1989; 63: 3362-3367Crossref PubMed Google Scholar, 34Ludlow J.W. Shon J. Pipas J.M. Livingston D.M. DeCaprio J.A. Cell. 1990; 60: 387-396Abstract Full Text PDF PubMed Scopus (292) Google Scholar, 35Peden K.W.C. Spence S.L. Tack L.C. Cartwright C.A. Srinivasan A. Pipas J.M. J. Virol. 1990; 64: 2912-2921Crossref PubMed Google Scholar, 36Lin H.-J.L. Upson R.H. Simmons D.T. J. Virol. 1992; 66: 5443-5452Crossref PubMed Google Scholar). The dl1135 mutant carries an in-frame deletion that results in the expression of a T antigen missing amino acids 17-27(29Pipas J.M. Peden K.W.C. Nathans D. Mol. Cell. Biol. 1983; 3: 203-213Crossref PubMed Scopus (118) Google Scholar). This mutant was of interest since it had been reported to be defective for both ATPase and sequence-specific DNA binding, activities thought not to require the amino-terminal domain(37Clark R. Peden K. Pipas J.M. Nathans D. Tjian R. Mol. Cell. Biol. 1983; 3: 220-228Crossref PubMed Scopus (58) Google Scholar). Additionally, dl1135 is defective for transformation of some cell types although it retains the ability to complex with pRb and p53. The mutation appears to abolish a third transforming activity of T antigen that can functionally replace the adenovirus E1A 300K binding function (38). To investigate in more detail the molecular basis for the in vivo replication defects of 5080 and dl1135, we expressed both of the mutant proteins using the baculovirus system and purified them by immunoaffinity chromatography(39Lanford R.E. Virology. 1988; 167: 72-81Crossref PubMed Scopus (105) Google Scholar, 40Murphy C.I. Weiner B. Bikel I. Piwnica-Worms H. Bradley M.K. Livingston D.M. J. Virol. 1988; 62: 2951-2959Crossref PubMed Google Scholar, 41O'Reilly D.R. Miller L.K. J. Virol. 1988; 62: 3109-3119Crossref PubMed Google Scholar). Purified baculovirus T antigen has been shown to be comparable to T antigen purified from infected monkey cells in all assays tested(42Hoss A. Moarefi I. Scheidtmann K.H. Cisek J.L. Corden J.L Donreiter I. Arthur A.K. Fanning E. J. Virol. 1990; 64: 4799-4807Crossref PubMed Google Scholar). Using the purified mutant proteins, we examined some of the biochemical activities of T antigen required for replication. Recombinant baculovirus 941T which expresses wild-type SV40 large T antigen was kindly provided by Robert Lanford(39Lanford R.E. Virology. 1988; 167: 72-81Crossref PubMed Scopus (105) Google Scholar). Both wild-type (wt) and mutant T antigens were expressed in BTI-TN-5B1-4 (“High 5”) insect cells (Invitrogen). The T antigen mutants dl1135, which has a deletion of amino acids 17-27, and 5080 which has an amino acid substitution of P584L have been described(28Peden K.W.C. Srinivasan A. Farber J.M. Pipas J.M. Virology. 1989; 168: 13-21Crossref PubMed Scopus (59) Google Scholar, 29Pipas J.M. Peden K.W.C. Nathans D. Mol. Cell. Biol. 1983; 3: 203-213Crossref PubMed Scopus (118) Google Scholar). The plasmids pdl1135 and p5080 contain the complete mutant viral genomes inserted at the BamHI site of pBR322 (28Peden K.W.C. Srinivasan A. Farber J.M. Pipas J.M. Virology. 1989; 168: 13-21Crossref PubMed Scopus (59) Google Scholar) or a derivative(29Pipas J.M. Peden K.W.C. Nathans D. Mol. Cell. Biol. 1983; 3: 203-213Crossref PubMed Scopus (118) Google Scholar). Recombinant baculovirus containing these mutants were constructed by digesting the pdl1135 plasmid or the p5080 plasmid with BamHI and StuI and ligating the purified SV40 fragment (2657 base pairs) into the BamHI and SmaI sites of the baculoviral transfer vector pVL1393. Subsequently, the recombinant transfer vectors containing the cDNAs of the mutants were made in an exchange reaction with 941T following digestion of both the wild-type and mutant constructs with EcoNI. The new recombinant transfer DNA was cotransfected with purified wild-type baculovirus DNA into insect cells followed by identification and purification of the recombinant virus by three rounds of dot-blot hybridization. High 5 insect cells were infected with the above recombinant baculoviruses, and the infected cells were lysed 36-42 h later using a buffer containing 0.2 M LiCl, 20 mM Tris, pH 8.0, 1 mM EDTA, 0.5% Nonidet P-40, 0.2 mM dithiothreitol (DTT),1( 1The abbreviations used are: DTTdithiothreitolAMP-PNPadenyl-5′-yl imidodiphosphate.) and a protease inhibitor mixture. The T antigens were immunoaffinity purified from the lysates using a protein A-Sepharose (Pharmacia) column cross-linked to either PAb416 (43) or PAb101 (44Gurney E.G. Tamowski S. Deppert W. J. Virol. 1986; 57: 1168-1172Crossref PubMed Google Scholar) as described previously(45Dixon R.A.F. Nathans D. J. Virol. 1985; 53: 1001-1004Crossref PubMed Google Scholar, 46Simanis V. Lane D.P. Virology. 1985; 144: 88-100Crossref PubMed Scopus (173) Google Scholar). After high pH elution (pH 11.4), the proteins were dialyzed overnight against a buffer containing 10 mM HEPES, pH 8.0, 1 mM EDTA, 0.1 M NaCl, 50% glycerol, 1 mM DTT, and 0.1 mM phenylmethylsulfonyl fluoride. dithiothreitol adenyl-5′-yl imidodiphosphate. pUC.HSO contains SV40 nucleotides 5171 to 128 (47). The pDV.XH plasmid carries the SV40 minimal origin of replication corresponding to 64 base pairs of sequence between SV40 nucleotides 5211 to 32(48Virshup D.M. Russo A.A. Kelly T.J. Mol. Cell. Biol. 1992; 12: 4883-4895Crossref PubMed Scopus (56) Google Scholar). pTBS1 contains T antigen binding site I within SV40 nucleotides 5171 to 5228(49Li J.J. Peden K.W.C. Dixon R.A.F. Kelly T.J. Mol. Cell. Biol. 1986; 6: 1117-1128Crossref PubMed Scopus (85) Google Scholar). The reactions were performed according to the methods previously described(47Wold M.S. Li J.J. Kelly T.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3643-3647Crossref PubMed Scopus (160) Google Scholar, 50Li J.J. Kelly T.J. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 6973-6977Crossref PubMed Scopus (353) Google Scholar). HeLa cell extracts, kindly provided by Dr. Thomas J. Kelly (Johns Hopkins University), were prepared as described previously(51Wobbe C.R. Dean F. Weissbach L. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5710-5714Crossref PubMed Scopus (228) Google Scholar). The DNA replication products were ethanol-precipitated, subjected to agarose gel electrophoresis, and visualized by autoradiography. Modified McKay2( 2R. A. F. Dixon, personal communication.) assays were done with site I and site II DNAs using two different buffers and conditions: McKay buffer and conditions and replication buffer with conditions appropriate for in vitro replication(52McKay R.D.G. J. Mol. Biol. 1981; 145: 471-488Crossref PubMed Scopus (121) Google Scholar). The McKay reaction buffer contained 10 mM HEPES, pH 7.3, 0.1 mM EDTA, 0.05% Nonidet P-40, 1 mg/ml bovine serum albumin, 2 mM DTT, and radiolabeled DNA. T antigen was then added, and the reactions were incubated on ice for 45 min to 1 h. Two hundred microliters of a 1:1 mixture of KT3/PAb101 hybridoma supernatant was added and the mixture was incubated for 30 min on ice, followed by the addition of 50 μl of protein A-Sepharose. The precipitated protein·DNA complex was washed three times with 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% Nonidet P-40. The replication buffer contained 30 mM HEPES, pH 7.5, 7 mM MgCl2, 1 mM DTT, 40 mM creatine phosphate, 10 mg/ml bovine serum albumin, plus radiolabeled DNA. These reactions were started by first adding T antigen and then 1 mM AMP-PNP (Sigma). The reactions were incubated at 37°C for 30 min, followed by the addition of 200 μl of KT3/PAb101 (44Gurney E.G. Tamowski S. Deppert W. J. Virol. 1986; 57: 1168-1172Crossref PubMed Google Scholar, 53MacArthur H. Walter G. J. Virol. 1984; 52: 483-491Crossref PubMed Google Scholar) and incubation for 20 min at 37°C. The protein A-Sepharose was then added and incubated for 15-20 min. The immunocomplex was washed with 1 M Tris-HCl, pH 7.5, 0.5 M EDTA, and 10% Nonidet P-40. In both instances, the final pellet was resuspended in 10 mM Tris-HCl, pH 7.5, 7.5 mM EDTA, 0.5% SDS, 10% glycerol and heated to 65°C for 2 min. The protein A-Sepharose beads were pelleted for 2 min, and the supernatant was analyzed by electrophoresis on a 1% or 1.4% agarose gel followed by autoradiography. The procedure has been described by Loeber et al.(22Loeber G. Stenger J.E. Ray S. Parsons R.E. Anderson M.E. Tegtmeyer P. J. Virol. 1991; 65: 3167-3174Crossref PubMed Google Scholar). T antigen was incubated at 37°C for 20-30 min in a reaction buffer consisting of 30 mM HEPES, pH 7.5, 7 mM MgCl2, 1 mM DTT, and 4 mM ATP. The protein was then cross-linked for 10-15 min at 37°C with 0.1% glutaraldehyde. Sample buffer containing 15% 0.5 M Tris-HCl, pH 6.8, 50% glycerol, and 0.0025% bromphenol blue at pH 6.8 was added, and the oligomers were analyzed on a 4-22% nondenaturing polyacrylamide gradient gel in Tris glycine buffer for 11 h at 4°C at 35 mA. The assay was performed as described by Virshup et al.(48Virshup D.M. Russo A.A. Kelly T.J. Mol. Cell. Biol. 1992; 12: 4883-4895Crossref PubMed Scopus (56) Google Scholar). A 67-base pair fragment containing the ori (site II) was obtained by digesting the pDV.XH plasmid with ApaLI and SnaBI, purified and radiolabeled. T antigen was added to an assay mixture containing HEPES, pH 7.8, 7 mM MgCl2, 40 mM creatine phosphate, 1 mM DTT with 4 mM AMP-PNP (Sigma), and 3 fmol (10,000-15,000 cpm/reaction) of 32P-labeled DNA fragment and the reactions were incubated at 37°C for 30 min after which the cross-linker glutaraldehyde was added to a final concentration of 0.05% and the reactions were incubated at 37°C for 5 more min. The DNA·protein complexes were electrophoresed on 0.5% agarose, 2.4% acrylamide gel in 89 mM Tris borate, 2 mM EDTA (TBE) for approximately 1 h at room temperature at 200 V. The gels were dried on DE81 Whatman paper and then exposed to XAR film at −70°C with an intensifying screen. The assay buffer consisted of 25 mM HEPES, pH 7.0, 5 mM MgCl2, 0.1 mM EDTA, 0.05% Nonidet P-40, 1 mM DTT, and 1.3 μM ATP including [α-32P]ATP. Each reaction mixture contained 10% glycerol. The reaction was started by the addition of T antigen and incubated at 37°C. At timed intervals, 1-8 μl of reaction mixture was pipetted into tubes containing an equal volume of 0.75 M KH2PO4, pH 3.5. Samples of 1-4 μl were subsequently spotted onto polyethyleneimine-cellulose plates and developed in 0.75 M KH2PO4 at room temperature for approximately 2 h. The plates were then exposed to XAR film, and the corresponding ATP and ADP spots were cut out and quantitated in a liquid scintillation counter, or the appropriate spots were quantitated by the AMBIS radioanalytic imaging system. This assay is based upon the method of Stahl et al.(54Stahl H. Droge P. Knippers R. EMBO J. 1986; 5: 1939-1944Crossref PubMed Scopus (235) Google Scholar). The annealed substrate consisting of M13mp18 (Life Technologies, Inc.) with a 17-nucleotide forward-sequencing primer (U. S. Biochemical Corp.) was extended from the primer using Klenow DNA polymerase in the presence of [α-32P]dATP, dGTP, and dTTP as described by Stahl et al.(54Stahl H. Droge P. Knippers R. EMBO J. 1986; 5: 1939-1944Crossref PubMed Scopus (235) Google Scholar). The assay mixture consisted of 0.3 fmol of annealed primer, 25 mM Tris, pH 7.5, 5 mM MgCl2, 2 mM ATP, 1 mM DTT, 40 mM phosphocreatine, 0.5 μg of creatine phosphokinase, 0.5 μg of poly(dI-dC) (Boehringer Mannheim). The reaction was started with the addition of 100 to 500 ng of T antigen followed by incubation at 37°C for 1 h. The reaction was stopped by the addition of an equal volume of 1.5% SDS, 0.2 M EDTA. Subsequently, 5 μl of sample buffer was added to each reaction, and the reactions were electrophoresed on a 12% acrylamide gel in TBE buffer for 2 h at 200 V. The unwinding assays were performed as described by Virshup et al.(55Virshup D.M. Kauffman M.G. Kelly T.J. EMBO J. 1989; 8: 3891-3898Crossref PubMed Scopus (70) Google Scholar). The plasmid pUC.HSO, containing the minimal origin of replication, was linearized with HindIII, labeled using the Klenow fragment and [α-32P]dCTP, and digested with BamHI and HinfI. This results in two labeled fragments, an ori+ and an ori- fragment. The ori- fragment served as an internal control for the unwinding reactions. T antigen was incubated with 0.5 fmol of labeled substrate DNA in a buffer containing 30 mM HEPES, pH 7.8, 7 mM MgCl2, 4 mM ATP, 40 mM creatine phosphate, and single-stranded DNA binding protein (United States Biochemical Corp.). The reactions were incubated for 1 h at 37°C and then stopped with 0.5% SDS, 10 mM Tris, pH 7.5, 5 mM EDTA, and proteinase K (10 mg/ml). The incubation was continued for 1 h at 37°C. DNA sample buffer was then added, and the reactions were heated at 65°C for 2 min. The reactions were then analyzed on an 8% acrylamide gel, dried on DE81 Whatman paper, and autoradiographed with XAR film. Recombinant baculoviruses expressing 5080 and dl1135 were constructed as described under “Experimental Procedures,” and the T antigens were subsequently immunoaffinity-purified from infected insect cells. Samples of each protein were assessed for purity and integrity by silver staining and Western blot analysis (Fig. 2). The silver stain shows a predominant band of approximately 97 kDa for both mutant and wild-type T antigens. Based on a visual assessment of this gel, we estimate that 5080 and dl1135 T antigens are greater than 90% pure. Fig. 2B shows a Western blot of purified dl1135 protein, and wild-type T antigen demonstrates that the monoclonal antibodies PAb101 and KT3, which recognize epitopes within the carboxyl-terminal region of T antigen, detect both mutant and wild-type proteins. However, the dl1135 deletion results in a loss of reactivity for PAb419 and PAb108. Since both PAb419 and PAb108 recognize denaturation-resistant epitopes, it seems likely that the deletion of residues 17-27 encompasses the actual epitopes for these monoclonal antibodies. The mutant protein 5080, like wild-type T antigen, is detected by antibodies PAb101 and KT3 as well as PAb419 and PAb108 (not shown). When electrophoresed through a nondenaturing gradient gel, T antigen resolves as multiple oligomeric forms that appear as a ladder extending from the monomer to dodecamer and higher. At 37°C in the presence of ATP and magnesium, T antigen undergoes a conformational change such that the predominant species is the hexamer(15Dean F.B. Dodson M. Echols H. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8981-8985Crossref PubMed Scopus (77) Google Scholar, 56Parsons R.E. Stenger J.E. Ray S. Welker R. Anderson M.E. Tegtmeyer P. J. Virol. 1991; 65: 2798-2806Crossref PubMed Google Scholar). This reaction can occur in the presence or absence of DNA; however, DNA cooperatively enhances protein to protein association(18Mastrangelo I.A. Hough P.V.C. Wall J.S. Dodson M. Dean F.B. Hurwitz J. Nature. 1989; 338: 658-662Crossref PubMed Scopus (263) Google Scholar, 56Parsons R.E. Stenger J.E. Ray S. Welker R. Anderson M.E. Tegtmeyer P. J. Virol. 1991; 65: 2798-2806Crossref PubMed Google Scholar). T antigen assembles on the origin of replication (ori) as a double hexamer in the presence of ATP and participates in localized melting of the origin(7Fanning E. Knippers R. Annu. Rev. Biochem. 1992; 61: 55-85Crossref PubMed Scopus (458) Google Scholar, 12Parsons R. Anderson M.E. Tegtmeyer P. J. Virol. 1990; 64: 509-518Crossref PubMed Google Scholar, 13Borowiec J.A. Hurwitz J. EMBO J. 1988; 7: 3149-3158Crossref PubMed Scopus (167) Google Scholar). Therefore, we wished to see if the mutant proteins formed the normal pattern of oligomeric forms and if they formed hexamers in response to ATP. The dl1135 protein resolved into an oligomeric ladder in the absence of ATP at 4°C without cross-linker, but unlike wild-type extended only from monomer to apparently hexamer with clear resolution. Some higher oligomeric forms were present, but these appeared fainter than with the wild-type T antigen (Fig. 3). There also seems to be a non-integer band with 1135 which is not present with wild-type, which we have seen in multiple experiments and for which we cannot account. When cross-linker was added at 4°C, the oligomeric forms appeared to be indistinguishable from wild-type. With the addition of 4 mM ATP at 37°C, some of the dl1135 protein shifted to the hexameric form, although most of it appeared to be in the monomeric form, suggesting that the response was less efficient than wild-type. Most of 5080 protein seems to be a smear in the gel with a only few lower molecular weight bands migrating near the level of dimer in the wild-type lane at 4°C without cross-linker (Fig. 3). With cross-linker, the few lower molecular weight bands are no longer visible, and there appears to be more smearing toward the top of the gel, suggesting more higher oligomeric or aggregated forms. The 5080 mutant also did not show any monomer as did both the wild-type and 1135 proteins. At 37°C with cross-linker in the presence and absence of ATP, the 5080 protein did not appear to enter the gel but remained at the top of the gel, suggesting that perhaps the protein forms a greater aggregate at the higher temperature or that the smaller oligomers are unstable at the higher temperature. This property of the 5080 T antigen has been reported previously by Lin et al.(36Lin H.-J.L. Upson R.H. Simmons D.T. J. Virol. 1992; 66: 5443-5452Crossref PubMed Google Scholar). We then examined the ability of the purified mutant T antigens expressed in the baculovirus system for sequence-specific DNA binding activity using an immunoprecipitation assay. A modified McKay assay was done using plasmids containing sites I or II in either a HEPES/EDTA buffer (McKay buffer) on ice or a HEPES/MgCl2 buffer with ATP and without EDTA (replication buffer) at 37°C. The wild-type T antigen appeared to bind to site I and site II slightly better in McKay buffer (on ice) than in replication buffer at 37°C (Fig. 4). The 5080 T antigen bound to site I nearly as well as wild-type in replication buffer. In contrast, 5080 bound to site I very inefficiently in McKay buffer, although it retained some specific DNA binding activity (Fig. 4) The 1135 protein also could specifically bind to site I, but the efficiency of the binding was much less than with wild-type. The 5080 T antigen bound very inefficiently to site II under both reaction conditions (Fig. 4). Site II binding by the 1135 protein was barely detectable when assayed in McKay buffer (Fig. 4). Clark et al.(37Clark R. Peden K. Pipas J.M. Nathans D. Tjian R. Mol. Cell. Biol. 1983; 3: 220-228Crossref PubMed Scopus (58) Google Scholar) also reported detecting no ori binding for the 1135 protein purified from infected CV1 monkey cells in a similar immunoprecipitation DNA binding assay. However, 1135 did bind to site II in replication buffer, but, in addition, it bound two other fragments. This binding is not entirely nonspecific since the fastest migrating band of 458 base pairs was not bound by 1135. This pattern of binding was seen in several independent experiments and with different protein preparations. We next wished to determine whether dl1135 and 5080 were capable of ATP-dependent assembly on the minimal origin (site II) of SV40 DNA. The ATP-dependent assembly of T antigen on the origin as a double hexamer is required for the presynthesis complex (for a review, see Ref. 7). Virshup et al.(55Virsh" @default.
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• W2007346089 title "T Antigens Encoded by Replication-defective Simian Virus 40 Mutants dl1135 and 5080" @default.
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