threonine 124 by cyclin-dependent kinases in controlling the origin unwinding activity of T antigen ... binds specifically to site II in the core origin, assembling in.
Vol. 67, No. 8
JOURNAL OF VIROLOGY, Aug. 1993, p. 4992-5002
0022-538X/93/084992-11$02.00/0 Copyright © 1993, American Society for Microbiology
Mutation of the Cyclin-Dependent Kinase Phosphorylation Site in Simian Virus 40 (SV40) Large T Antigen Specifically Blocks SV40 Origin DNA Unwinding ISMAIL F. MOAREFI,1 DON SMALL,2 ILKA GILBERT,' MATTHIAS HOPFNER,1 SANDRA K. RANDALL,2 CHRISTINE SCHNEIDER,' ALICIA A. R. RUSSO,2 UWE RAMSPERGER,3 AVRIL K. ARTHUR,' HANS STAHL,3 THOMAS J. KELLY,2 AND ELLEN FANNING'*
Institute for Biochemistry, Karlstrasse 23, D-8000 Munich 2,1 and Fakultat fisr Biologie, Universitat Konstanz, D-7750 Konstanz, Gennany, and Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 212052 Received 15 March 1993/Accepted 6 May 1993
A mutant simian virus 40 (SV40) large tumor (T) antigen bearing alanine instead of threonine at residue 124 (T124A) failed to replicate SV40 DNA in infected monkey cells (J. Schneider and E. Fanning, J. Virol. 62:1598-1605, 1988). We investigated the biochemical properties of T124A T antigen in greater detail by using purified protein from a baculovirus expression system. Purified T124A is defective in SV40 DNA replication in vitro, but does bind specifically to the viral origin under the conditions normally used for DNA replication. The mutant protein forms double-hexamer complexes at the origin in an ATP-dependent fashion, although the binding reaction requires somewhat higher protein concentrations than the wild-type protein. Binding of T124A protein results in local distortion of the origin DNA similar to that observed with the wild-type protein. These findings indicate that the replication defect of T124A protein is not due to failure to recognize and occupy the origin. Under some conditions T124A is capable of unwinding short origin DNA fragments. However, the mutant protein is almost completely defective in unwinding of circular plasmid DNA molecules containing the SV40 origin. Since the helicase activity of T124A is essentially identical to that of the wild-type protein, we conclude that the mutant is defective in the initial opening of the duplex at the origin, possibly as a result of altered hexamer-hexamer interactions. The phenotype of T124A suggests a possible role for phosphorylation of threonine 124 by cyclin-dependent kinases in controlling the origin unwinding activity of T antigen in infected cells.
origin: melting in the early palindrome and untwisting in the A+T-rich region (4, 5, 16, 19, 61, 62, 68). Protein-protein interactions between T antigen, DNA polymerase a-primase, and replication protein A (RP-A) are thought to recruit these key initiation proteins to the nucleoprotein complex on the origin and facilitate the synthesis of the first primers (15, 27-29, 34, 35, 54, 56a, 58a, 75). In the presence of the single-strand DNA-binding protein RP-A, the DNA helicase activity of T antigen unwinds the template DNA more extensively (8, 9, 17, 26, 78, 93). After synthesis of the first Okazaki fragments by polymerase a-primase, DNA polymerase 8 and its auxiliary factors assemble at the primertemplate junction to form the leading strand replication complex, while polymerase a-primase becomes the lagging strand replicase (85-87). A possible role for a second lagging strand polymerase complex has also been suggested (9, 59, 60). Several lines of evidence indicate that the replication initiation activity of T antigen depends on its state of phosphorylation (reviewed in references 32 and 65). Phosphorylation on several serine residues appears to downregulate the replication activity of T antigen, while phosphorylation on threonine 124 (Thr-124) is required. T antigen can be specifically phosphorylated on Thr-124 by cdc2-related cyclin-dependent protein kinases (42, 56). Bacterially produced T antigen was unable to initiate SV40 DNA replication in vitro unless it was modified at Thr-124 by a cdc2-related kinase (56). A mutant T antigen carrying alanine in place of Thr-124 (T124A) also failed to replicate SV40 DNA in vitro and was defective in its ability to initiate replication in vitro
Knowledge of eucaryotic DNA replication has been advanced significantly by studies with simian virus 40 (SV40) DNA replication as a model system (reviewed in references 3, 11, 23, 45, and 79). Replication occurs only during the S phase of the cell cycle and is dependent on cellular replication proteins, except for the multifunctional viral protein large tumor (T) antigen (reviewed in references 32, 33, and 65). Purification of the cellular proteins required for SV40 DNA replication has been greatly facilitated by a cell-free SV40 DNA replication system, originally developed by Li and Kelly (51), that closely resembles SV40 replication in the cell. Using purified cellular proteins and T antigen, SV40 replication can be reconstituted in vitro, suggesting that most of the essential cellular proteins are now known (46, 85, 91). Initiation of SV40 DNA replication takes place in a series of steps that can be defined in partial reactions with purified proteins in vitro. The complete SV40 origin of replication is composed of a 64-bp core origin required for replication and two adjacent auxiliary regions that enhance replication: the Spl binding sites on the late side of the core, and the T-antigen binding site I on the early side (reviewed in references 3 and 22). In the first step of initiation, T antigen binds specifically to site II in the core origin, assembling in the presence of ATP into a double hexamer on the origin (6, 16, 18, 20, 26, 53, 63). Assembly of this nucleoprotein complex results in local conformation changes in the core *
Corresponding author. 4992
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MUTANT SV40 T ANTIGEN DEFECTIVE IN ORIGIN UNWINDING
(30). Moreover, the T124A T antigen did not replicate SV40 DNA in monkey cells in culture (74). An immunoprecipitation DNA binding assay demonstrated that T124A and bacterial T antigen not phosphorylated at residue 124 failed to bind to site II in the core origin DNA (56, 74), suggesting that it was a defect in origin DNA binding activity that led to loss of DNA replication activity. We demonstrate in the present report that under DNA replication conditions, the T124A mutant protein is able to bind to the SV40 origin, assemble into a double hexamer at the origin, and generate local conformational distortions in the origin DNA. However, the mutant T antigen cannot activate unwinding of relaxed closed circular or supercoiled origin DNA under replication conditions. A new role for phosphorylation of Thr-124 by cyclin-dependent kinases in regulation of SV40 origin DNA unwinding is discussed. (Preliminary reports on these studies were presented at the EMBO Workshop on Molecular Biology of DNA Replication in Weggis, Switzerland, 6-11 September, 1992 and at the DNA Tumor Virus Meeting in Cold Spring Harbor, 12-16 August, 1992.) MATERIALS AND METHODS T antigens. Recombinant baculoviruses were used to express wild-type (wt) (49) and T124A mutant (57) T antigens in Sf9 insect cells. T antigens were isolated by immunoaffinity chromatography as described previously (42). T antigens were eluted from the antibody matrix in 20 mM triethanolamine (pH 10.8)-20% glycerol and neutralized with 0.1 volume of 0.5 M PIPES [piperazine-N,N'-bis(2-ethanesulfonic acid)]-KOH (pH 7.0). The concentrations of T antigens were usually 0.8 to 0.9 mg/ml for wt and 0.6 to 0.8 mg/ml for T124A as determined spectrophotometrically (37). T antigens stored in aliquots at -85°C were thawed on ice, used once, and then discarded. Plasmid DNAs. pONwt carries a 19-bp synthetic oligonucleotide sequence comprising T antigen binding site I (71). p1097 carries an SV40 genome with a 31-bp deletion encompassing all of site I (25, 74). pOR1 contains SV40 nucleotides 5171 to 37 with T antigen binding site I deleted (21). pUC-HS contains SV40 nucleotides 5171 to 128 (83); pUC.HSO contains SV40 nucleotides 5171 to 128 (93). In vitro SV40 DNA replication reactions. Replication reactions were done by the methods of Guo et al. (40) or Wold et al. (93) with similar results. S100 cytoplasmic extracts were prepared from human 293 and Raji cells as described previously (52). The replication reaction contained 150 ng of pUC-HS DNA, 190 p,g of S100 extract, 30 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-KOH (pH 7.8), 0.5 mM dithiothreitol, 7 mM Mg acetate, 1 mM EGTA (pH 8), 4 mM ATP, 0.08 mM CTP, GTP, and UTP, 0.1 mM dATP and dGTP, 0.04 mM dCTP and dTI7P, 5 ,Ci each of [a-32P]dC1TP and [a-32P]d1TTFP (3,000 Ci/mmol), 20 mM phosphoenolpyruvate (PEP), 1.6 ,ug of pyruvate kinase, and 0 to 600 ng of T antigen in a volume of 60 ,ul (40). Alternatively, replication reactions contained 100 ng of pUC.HSO DNA, 100 ,ug of S100 extract, 30 mM HEPES-KOH (pH 7.5), 25 mM Na phosphate, 7 mM MgCl2, 4 mM ATP, 0.2 mM CTP, GTP, and UTP, 0.1 mM dGTP, dATP, and dTFITP, 0.05 mM dCTP, 25 p,Ci of [a-32P]dCTP (3,000 Ci/mmol), 40 mM creatine phosphate, 100 ,ug of creatine kinase, and 0 to 800 ng of T antigen in a volume of 25 RI (93). Immunoprecipitation DNA binding assay. A mixture of S ,g of EcoRI-HindIll-digested p1097 DNA (contains no T
4993
antigen binding site I [25]) and 2.5 ,ug of EcoRI-SalI-digested pONwt DNA (contains site I only) was 5' end labeled with T4 polynucleotide kinase and [-y- 2P]ATP (3,000 Ci/mmol) (74). A 0.09-,ug sample of the mixed fragments was assembled with different amounts of T antigen and 500 ng of purified monoclonal antibody Pab416 (41) and adjusted to the conditions specified in the legend to Fig. 3. Immune complexes and bound DNA were precipitated with formalinfixed Staphylococcus aureus, washed three times at 4°C with 50 mM Tris-HCl (pH 7.5)-5 mM EDTA-0.05% Nonidet P-40 (NET), with or without 150 mM NaCl, resuspended in 125 mM Tris-HCl (pH 8.3)-20% glycerol-10% P-mercaptoethanol-4% sodium dodecyl sulfate (SDS), and heated at 65°C for 10 min. After the bacteria were pelleted, the supernatant was analyzed by agarose gel electrophoresis and autoradiography of the dried gel. Mobility shift DNA binding assay. ATP-dependent assembly of T antigen complexes on SV40 core origin DNA was done essentially as described previously (18) with pOR1 DNA (21) digested with PvuI and Hinfl. Reactions contained 0.5 ,ug of DNA fragments, 30 mM HEPES-KOH (pH 7.8), 0.5 mM dithiothreitol, 7 mM MgCl2, 4 mM ATP, 20 mM PEP, 375 ng of pyruvate kinase, and 0 to 1 ,g of T antigen in a volume of 15 ,ul. After 30 min at 37°C, protein-DNA complexes were cross-linked with 0.1% glutaraldehyde at 37°C for 15 min. Samples were adjusted to 4% sucrose, 15 mM EDTA, and 0.02% bromphenol blue and electrophoresed in 1.5% agarose gels in 25 mM Tris-HCl-38 mM glycine0.2 mM EDTA (pH 8.5) at 40 V for 3 to 4 h. Protein-DNA complexes and free DNA were visualized by staining with ethidium bromide and photographed under UV light. KMnO4 footprinting. Reaction mixtures (30 pl) contained 0.5 ,ug of pUC-HS DNA, 30 mM HEPES-KOH (pH 7.8), 0.5 mM dithiothreitol, 7 mM MgCl2, 4 mM ATP, 20 mM PEP, 0.8 ,ug of pyruvate kinase, and 1.5 ,g of T antigen. After incubation of the mixtures for 30 min at 37°C, KMnO4 was added to give a final concentration of 30 mM KMnO4. The reaction mixture was further incubated at 37°C for 3 min. The reaction was stopped by the addition of 3-mercaptoethanol to 1.0 M. Samples were phenol-chloroform extracted twice and subsequently desalted with Quick Spin columns (Boehringer Mannheim) preequilibrated in water. To detect modifications, a reverse sequencing primer (Pharmacia-PL, Freiburg, Germany) was 5' end labeled with 2p04, using T4 polynucleotide kinase (Pharmacia-PL) and used for primer extension by the method of Borowiec et al. (7). Sequence positions of modified residues were determined by comparison with dideoxy sequencing ladders. DNA helicase assay. To prepare substrate, M13mp9 DNA (Boehringer Mannheim) was annealed with a 17-nucleotide forward-sequencing primer (Pharmacia-PL) which was subseqXuently 3' end labeled with Klenow DNA polymerase with [a- 2P]dATP and unlabeled dGTP to extend the primer by three nucleotides. Reaction mixtures contained 5 ng of substrate, 30 mM HEPES-KOH (pH 7.8), 1 mM dithiothreitol, 7 mM Mg acetate, 1 mM EGTA, 4 mM ATP, 20 mM PEP, and 0 to 6 ,ug of T antigen in a volume of 20 RlI. After incubation of the mixtures at 37°C for 30 min, 10 pl of 0.625 M Tris-HCl (pH 6.8)-100 mM dithiothreitol-0.05% bromphenol blue-25% glycerol was added, and the samples were loaded immediately onto 8% nondenaturing polyacrylamide gels and electrophoresed. Helicase activity was visualized by autoradiography of the dried gels. Origin DNA unwinding assay. Reaction mixtures contained 200 ng of predominantly supercoiled or relaxed closed circular pUC-HS DNA, 30 mM HEPES-KOH (pH 7.8), 1
4994
MOAREFI ET AL.
mM dithiothreitol, 1 mM EGTA, 7 mM Mg acetate, 4 mM ATP, 20 mM PEP, 1.6 ,ug of pyruvate kinase, 250 ng of Escherichia coli SSB (Pharmacia-PL), 10 ,ug of bovine serum albumin, 0.7 ,ul of topoisomerase I, and different amounts of wt or T124A T antigen in a volume of 40 pl (47). After 1 h at 37°C, the DNA was first deproteinized by incubation in 0.4% SDS-400 ng proteinase K at 37°C for 30 min and then ethanol precipitated. The samples were redissolved in 10 mM EDTA-2% Ficoll-2% sucrose-0.01% bromphenol blue0.1% SDS and electrophoresed in 1.2% agarose gels (47). Topoisomerase I was partially purified from calf thymus nuclei by hydroxylapatite and phenyl-Sepharose chromatography as described previously (81). An amount of this fraction suitable for the unwinding assay was determined empirically. Electron microscopy. Unwinding reaction mixtures under replication conditions contained 100 ng of supercoiled pUC-HS DNA, 30 mM HEPES-KOH (pH 7.8), 1 mM dithiothreitol, 7 mM MgCl2, 4 mM ATP, 40 mM creatine phosphate, 0.1 mg of creatine kinase per ml, 25 mM NaPO4 (pH 7.2), 5 U of wheat germ topoisomerase I (Promega, Madison, Wis.), and 200 ng of E. coli SSB (Stratagene, La Jolla, Calif.). The mixtures were incubated for 15 min at 37°C to relax supercoiled DNA. wt or T124A T antigen was then added at 2 ,ug to a final reaction volume of 25 ,ul and further incubated for 1 h at 37°C. DNA-protein complexes were cross-linked with 0.2% glutaraldehyde for 15 min at 37°C and purified with a 1-ml syringe column of Sepharose CL-4B (Pharmacia-LKB, Piscataway, N.J.) equilibrated in 40 mM HEPES (pH 7.8)-11 mM Mg acetate-0.01% glutaraldehyde. The DNA-protein complexes were spread on 400-mesh carbon-coated grids with benzyldimethylalkylammonium chloride, stained with 50 ,uM uranyl acetate, shadowed with Pt-Pd, and visualized at x170,000 with a JEM-100CX electron microscope (13). More than 2,000 mutant nucleoprotein complexes were analyzed. Unlabeled nucleoside triphosphates were purchased from Pharmacia-PL, and radiochemicals were from Amersham (Braunschweig, Germany). Sources of enzymes not specified above were New England BioLabs (Schwalbach, Germany), Boehringer (Mannheim, Germany), or PharmaciaPL.
J. VIROL.
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FIG. 1. Purification of baculovirus-expressed wt and mutant T antigens. Immunoaffinity-purified wt and T124A mutant T antigens (1 jig) were electrophoresed in denaturing polyacrylamide gels (48) and then stained with Coomassie blue. The molecular weights of prestained marker proteins (Sigma Chemical Co., Deisenhofen, Germany) are shown at the left in kilodaltons.
pUC.HSO plasmid DNA bearing the complete SV40 origin of replication on a HindIII-SphI fragment (93). Incorporation of labeled deoxyribonucleotides into ethanol-precipitable material increased as a function of the amount of wt T antigen present in the reaction (Fig. 2). However, little or no replication activity of T124A T antigen was detected. Even when the level of T124A in the reaction mixture was increased to 2 jig, synthesis of SV40 DNA was less than 15% of that observed with the wt protein (30, 70). SV40 DNA-binding activity of T124A T antigen. Initial studies of the origin DNA-binding activity of T124A T antigen expressed in transformed rat cells indicated that its ability to bind to site II in the origin of replication was defective, while its ability to bind to site I remained intact (74), suggesting that impaired site II DNA binding could be the source of the replication defect. These experiments were repeated with purified T antigens expressed in the baculovirus system. The wt T antigen prepared from this system was shown to be essentially quantitatively phosphorylated T Antigen wt . p,p I~
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RESULTS SV40 DNA replication activity of T124A T antigen. Initial characterization of the T124A mutant T antigen demonstrated that it failed to direct viral DNA replication in monkey cells in culture (74). To ascertain whether the mutant was defective in the replication process itself or in events prior to but required for replication of viral DNA in monkey cells, we wished to assess its DNA replication activity in vitro. To this end, a recombinant baculovirus was constructed to express the mutant T antigen in infected insect cells (57). T124A and wt T antigens were purified by immunoaffinity chromatography, and samples of each were characterized by electrophoresis in a denaturing polyacrylamide gel and then by staining with Coomassie blue (Fig. 1). A major band of monomeric denatured T antigen and a minor band of more slowly migrating incompletely denatured T antigen were detected. Both bands reacted with anti-T monoclonal antibodies in immunoblots (data not shown). The SV40 DNA replication activity of purified T124A T antigen was assayed in a cell-free system (40, 51, 80) with wt T antigen as a control. The template DNA was supercoiled
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FIG. 2. SV40 DNA replication activity of wt and T124A T antigens in human cell extracts. Replication reactions with pUC.HSO DNA and the indicated amount of T antigen were done by the method of Wold et al. (93) as described in Materials and Methods. (A) After 2 h at 37°C, reactions were stopped by the addition of SDS and proteinase K; the replication products were precipitated, redissolved, and analyzed by electrophoresis in 1% agarose gels and autoradiography. (B) Replication was quantitated by counting the radioactivity in an aliquot of each reaction after acid precipitation.
VOL. 67, 1993
MUTANT SV40 T ANTIGEN DEFECTIVE IN ORIGIN UNWINDING
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FIG. 3. SV40 DNA-binding activity of wt and T124A T antigens is similar under replication conditions. A mixture of end-labeled DNA fragments containing T-antigen-binding site I or II or nonspecific DNA (0.09 p,g) was incubated together with the indicated amounts of T antigen and excess purified Pab 416 monoclonal antibody for 30 min at 37°C. In the panels on the right, reaction mixtures contained 30 mM HEPES-KOH (pH 7.8), 1 mM dithiothreitol, 7 mM Mg acetate, 1 mM EGTA, 4 mM ATP, 20 mM PEP, 10 pg of bovine serum albumin, and 1.6 p.g of pyruvate kinase. In the panels on the left, reaction mixtures contained 30 mM HEPES-KOH (pH 7.8), 1 mM dithiothreitol, 10 pg of bovine serum albumin, and 80 mM KCI. T antigen-antibody-DNA complexes were precipitated by the addition of fixed S. aureus for 10 min at 37C. After washing with NET (left) or NET without NaCl (right), bound DNA was dissociated from the complexes and analyzed by agarose gel electrophoresis and autoradiography (A). A sample of the DNA fragment mixture served as a marker. Densitometry of the autoradiograms was used to quantitatively compare the DNA-binding activities of wt and mutant proteins (B and C). AU, arbitrary units.
on Thr-124 and underphosphorylated on serine residues (42). Immunoprecipitation of labeled DNA fragments bound to T124A or wt T antigen was performed under the conditions used previously (Fig. 3, left). In the presence of 80 mM KCI and without Mg-ATP, T124A T antigen bound normally to site I, but poorly to site II compared with wt T antigen (Fig. 3, left), as described previously (74). However, since these conditions are incompatible with the replication activity of wt T antigen, DNA binding studies under these conditions may not be relevant to viral DNA replication activity. Thus, we reexamined the SV40 DNA-binding activity of T124A T antigen under conditions similar to those used for replication assays. Under these conditions, specific binding of T124A T antigen to site I and to site II was comparable to that of wt T antigen (Fig. 3, right). This result rules out impaired site II DNA binding of T124A as the cause of its replication defect. The SV40 DNA-binding activity of T124A T antigen was further investigated by a DNA mobility shift assay originally developed to detect the ATP-dependent assembly of T-antigen double hexamers on the core origin and on the complete origin of replication (18, 26, 53, 90). Various amounts of T124A or wt T antigen were incubated under replication conditions with a mixture of DNA fragments, the largest of
which harbored the core origin of replication. The core origin (pOR1) rather than complete origin was chosen for these experiments to ensure that any site II DNA binding observed would be unaffected by site I DNA binding of T antigen. DNA-protein complexes were cross-linked with glutaraldehyde, the reaction products were separated by electrophoresis in a nondenaturing agarose gel, and the DNA was visualized by ethidium bromide staining. The DNA fragment bearing the core origin of replication was shifted to a lower mobility in the presence of as little as 0.1 to 0.25 ,ig of wt T antigen (Fig. 4A, lanes 2 to 5; data not shown). The mobility shift was dependent on the presence of ATP or dATP and on the presence of the origin sequences, as reported previously (18). The mobility shift was observed when the reaction was started by addition of T antigen or ATP. However, when the DNA was added last to the reaction mixture, no shift occurred, in agreement with previous reports that preassembled hexamers are unable to bind to origin DNA (16, 53). A DNA mobility shift was also detected with T124A T antigen (Fig. 4A, lanes 8 to 10). Complex formation with T124A was first observed with 0.5 Fg of T antigen, resulting in a band of intermediate mobility which probably contains a
4996
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J. VIROL.
MOAREFI ET AL.
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4. Assembly of T-antigen multimers on SV40 core origin (A) pORi DNA fragments were incubated with the indicated amounts of wt or T124A T antigen (TAg) under replication conditions. Protein-DNA complexes were cross-linked with glutaraldehyde and analyzed by electrophoresis in agarose gels and ethidium bromide staining. Identical results were obtained with creatine phosphate and creatine kinase as the ATP-regenerating system (data not shown). (B) Supercoiled pUC-HS DNA was incubated with wt or T124A T antigen under replication conditions. After 30 min at 370C, T antigen-induced structural changes in the origin region were detected by permanganate footprinting (lanes 5 to 8). Lanes 1 to 4 show DNA-sequencing reactions; note that the A lane corresponds to Ts oxidized by KMnO4. The early palindrome (EP), T antigenbinding site (TA g), and the A+T-rich region of the SV40 origin
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single hexamer of T antigen (Fig. 4A, lane 8) (90). The full mobility shift corresponding to double-hexamer assembly required approximately twice as much T124A as wt T antigen to achieve a comparable amount of origin-bound double hexamer (Fig. 4A, compare lane 9 with lanes 2 and 3). Even the largest amount of mutant T antigen was unable to shift all of the origin-containing DNA fragment to the fully complexed form (Fig. 4A, lane 10). These results indicate that T124A is competent to assemble on the core origin into double-hexamer complexes, although the reaction is less cooperative and the apparent overall affinity of T124A for the origin may be reduced. At high T-antigen concentrations, most of the origin DNA was driven into double-hexamer
complexes by both mutant and wt protein. Since at these concentrations the T124A T antigen fails to support significant SV40 DNA replication in vitro, the replication defect cannot be due simply to failure to bind to the origin. Specific DNA binding of T antigen and double-hexamer assembly at site II in the SV40 origin of replication leads to local DNA melting in the early palindrome and untwisting in the A+T-rich region of the core origin (reviewed in reference 3; 4, 16, 19, 62). These local conformational alterations can be detected by chemical probing of the distorted sequences (7). Thus, KMnO4 footprinting was performed on wt and T124A T antigen-origin DNA complexes assembled under replication conditions. With the complete origin DNA (pUCHS) as supercoiled template, binding of wt T antigen induced one major and two minor KMnO4-sensitive bands in the early palindrome and two sites in the A+T-rich region (Fig. 4B, lanes 5 and 6). An identical pattern of KMnO4 sensitivity was observed with T124A T antigen (lanes 7 and 8). The oxidized bands corresponded to nucleotides 5217/5218, 5214, and 5210 in the early palindrome and 20 and 29/30 in the A+T-rich region, in agreement with previous reports (5, 61). These results suggest that binding of T124A T antigen to site II in the SV40 origin produces the same local structural distortions in the flanking sequences as does wt T antigen. DNA helicase activity of T124A T antigen. The finding that the replication defect of T124A cannot be explained by a failure of the protein to bind to the origin raised the question whether a later step in the initiation of replication might be impaired in this mutant. Initial studies of T124A T antigen had demonstrated an ATPase activity comparable to that of wt (74). However, defective DNA helicase activity in the mutant remained a possibility. Thus, we compared the helicase activities of wt and T124A T antigens using a partially double-stranded DNA as the substrate. The activity of the mutant resembled that of wt T antigen at all concentrations except the lowest one tested (Fig. 5). Similar results were observed with several different helicase substrates and assay conditions (77). We conclude that the DNA helicase activity of T124A T antigen is intact. Origin DNA-unwinding activity of T124A T antigen. During initiation of SV40 replication, T antigen first locally distorts the origin DNA and then in an ATPase-dependent reaction progressively unwinds it in the presence of RP-A (reviewed in reference 3). Several assays have been developed to assay origin unwinding; the requirement for RP-A in these assays can also be satisfied by using E. coli SSB (17, 93). The origin-unwinding activity of T124A T antigen was initially investigated with an end-labeled double-stranded core origin DNA fragment as the substrate or, as a control for origin specificity, a fragment of similar size carrying only bacterial plasmid sequences (38, 90). Under replication conditions, both wt and T124A T antigen unwound the origin-containing fragment specifically, generating labeled single-stranded DNA of lower electrophoretic mobility, while most of the nonspecific control DNA remained double stranded (Fig. 6A). The unwinding activity of T124A was clearly lower than that of wt T antigen, but this difference is much smaller than the difference in the replication activities of the two proteins (compare Fig. 6B and Fig. 2). Similar results were obtained with a 311-bp double-stranded fragment containing the complete SV40 origin. The results suggest that with small DNA fragments as the substrate, the origin-specific unwinding activity of T124A T antigen is nearly comparable to that of wt T antigen. Since the natural template for SV40 DNA replication is a circular duplex DNA molecule, we examined the ability of
MUTANT SV40 T ANTIGEN DEFECTIVE IN ORIGIN UNWINDING
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FIG. 5. DNA helicase activity of T124A T antigen is similar to that of wt T antigen. Helicase reactions were done under replication conditions and contained 5 ng of single-stranded M13 DNA annealed to an end-labeled oligonucleotide and the indicated amounts of T antigen. Reaction products were analyzed by electrophoresis in nondenaturing polyacrylamide gels and autoradiography (left). Autoradiograms were evaluated by microdensitometry (right).
T124A T antigen to support unwinding of such templates in reactions containing topoisomerase I and E. coli SSB (17, 26, 93). Electron microscopy indicated that T124A readily bound to the template as expected (Fig. 7B). Length measurements of molecules linearized at a unique restriction site demonstrated that the majority of the T124A complexes
A
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FIG. 6. Specific unwinding of a double-stranded SV40 origin DNA fragment under replication conditions. The substrate for the unwinding reaction consisted of 0.3 fmol each of two 32P-labeled fragments, one containing an SV40 core origin (ori) and the other of similar size but containing only plasmid sequences (ns). The DNA was incubated with an increasing amount of wt and T124A T antigens in a final volume of a 10-J.l mixture containing 81 ng of E. coli SSB, 25 mM NaPO4 (pH 7.5), 30 mM HEPES (pH 7.5), 7 mM MgCl2, 4 mM ATP, 0.5 mM dithiothreitol, 40 mM creatine P04, and 100 ng of creatine kinase. After 30 min at 37°C, the samples were digested with SDS-proteinase K and electrophoresed in 8% polyacrylamide gels which were then dried and exposed on film. (A) Autoradiogram. Lane 14 shows a control reaction with substrates but without T antigen. Lane 13 shows the boiled substrates as a control. (B) Amount of unwinding in each lane of a gel from a similar experiment was quantitated with a Phosphorimager.
FIG. 7. T124A T antigen can bind specifically but not unwind a relaxed closed circular SV40 origin-containing plasmid under replication conditions. Unwinding reactions under replication conditions and processing for electron microscopy were as described in Materials and Methods. (A) Typical unwound intermediate generated by wt T antigen (about 20% of the nucleoprotein complexes). (B) Unwinding intermediates were generated by T124A T antigen at
