JOURNAL OF VIROLOGY, OCt. 1990, p. 4820-4829
Vol. 64, No. 10
0022-538X/90/104820-10$02.00/0 Copyright © 1990, American Society for Microbiology
Assembly of Nascent DNA into Nucleosome Structures in Simian Virus 40 Chromosomes by HeLa Cell Extract KAORU SUGASAWA,12t* YASUFUMI MURAKAMI,3 NORIMASA MIYAMOTO,3 FUMIO HANAOKA,"12 AND MICHIO UI1 Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113,1 Radiation Biology Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-01,2 and Tsukuba Life Science Research Center, RIKEN, Tsukuba, Ibaraki 305,3 Japan Received 2 February 1990/Accepted 9 July 1990
A soluble system was developed that could support DNA replication in simian virus 40 (SV40) chromosomes. DNA synthesis in this system required the presence of purified SV40 large tumor antigen, SV40 chromosomes prepared from virus-infected monkey cells, a crude extract from HeLa cells, and several low-molecular-weight components. In comparison to the replication of purified SV40 form I DNA, the rate of DNA synthesis was 15 to 20% in this system. DNA synthesis started near the replication origin of SV40 and proceeded bidirectionally in a semiconservative manner. Micrococcal nuclease digestion experiments revealed that the replicated DNA produced in this system became organized into a regularly spaced array of nucleosome core particles when an appropriate amount of purified HeLa core histones was added to the reaction mixture. SV40 form I DNA replicating under the same conditions was also assembled into nucleosomes, which were arranged in a rather dispersed manner and formed an aberrant chromatin structure.
In eucaryotic cells, the protein structure of chromosomes as well as DNA must be duplicated during the S phase of the cell division cycle. A number of observations have suggested that gene expression may somehow be regulated by the higher-order chromatin structure of each gene (reviewed in references 31 and 33). Although it is still uncertain whether chromatin structure actually plays any intrinsic role in gene regulation, it is possible that the events of DNA metabolism other than transcription, such as replication, recombination, and repair, may also be affected by the chromatin structure. Moreover, it has been suggested that some features of the chromatin structure, which influence the transcriptional activity of genes, may be stably propagated during cell proliferation to transmit a pattern of gene expression from parent to progeny cells (3, 8, 51, 52). Despite many elaborately designed experiments, however, the mechanisms involved in the propagation of chromatin structure in vivo remain poorly understood. For instance, even the modes of segregation and duplication of the most basic protein structure, the histone octamer, remain controversial. This is possibly because of the heterogeneity and intractability of the chromatin structure in intact cells (1, 4, 7, 12-16, 20-22, 32, 35-37, 39, 41, 45,
48). Under these circumstances, it was thought to be useful to develop a simple cell-free system, in which not only DNA but also its chromatin structure could be duplicated. Several cell-free systems have been described that can support faithful initiation and elongation in the replication of simian virus 40 (SV40) DNA or plasmid DNA containing SV40 ori. These systems used cell extracts from monkey cells (24) or human cells (25, 44, 53) fortified with separately purified SV40 T antigen as the source of replication proteins. Extensive biochemical and genetic analyses of these systems are underway to elucidate the molecular mechanisms involved
in the replication of viral DNA per se (reviewed in reference 17). More recently, Ishimi et al. (10) reported the reconstitution of the replication reactions of SV40 ori-containing plasmid DNA by using only purified enzymes and factors from HeLa cells plus the viral T antigen. However, the role of the chromatin structure has been little considered in these studies. Stillman (43) reported that the selective assembly of replicating SV40 DNA into a chromatin structure could occur with the addition to his reaction system of a nuclear extract from human 293 cells. More recently, Smith and Stillman (42) have reported the purification and characterization of a cellular factor required for replication-dependent chromatin assembly (CAF-I), although the template DNA used in their studies was purified to be nonnucleosomal. In addition, Decker et al. (5) reported a soluble system containing cytosol and nuclear extract fractions prepared from SV40-infected cells, which could initiate DNA replication in endogenous SV40 chromosomes, but the chromatin assembly of the newly synthesized DNA was not examined by these workers. In this paper, we describe the construction of a new soluble system that can support the DNA replication of SV40 chromosomes. This system contains SV40 chromosomes prepared from virus-infected CV-1 cells, a crude extract from uninfected HeLa cells, immunoaffinity-purified SV40 T antigen, and several other materials. The mode of viral DNA replication in this system mimicked that seen in vivo with respect to dependence on T antigen, bidirectional and semiconservative synthesis, and several other factors. Furthermore, digestion experiments with micrococcal nuclease strongly suggested that the newly synthesized DNA was assembled into a regularly spaced array of nucleosomes and that duplication of the nucleosome core structure must therefore occur in this reaction system.
Corresponding author. t Present address: Radiation Biology Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-01, Japan.
MATERIALS AND METHODS Extracts, T antigens, and DNA. Crude extracts from HeLa cells grown in suspension and SV40 large T antigen were prepared according to the procedures of Wobbe et al. (53).
*
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VOL. 64, 1990
Extracts from mouse mammary carcinoma FM3A cells were prepared in the same way as the extracts from HeLa cells. Polyomavirus large T antigen was produced in an insect cell line, SF27 cells infected with a recombinant baculovirus, vEV51LT (34). It was then purified by immunoaffinity chromatography by using a monoclonal antibody against polyomavirus large T antigen, F4 (30), as described by Murakami et al. (27). The construction of plasmid pBE102, which contains the replication origin of polyomavirus, has been described by Kern et al. (18). SV40 form I DNA was purchased from Bethesda Research Laboratories, Inc. Preparation of SV40 chromosomes. The SV40 chromosomal fraction was prepared on the basis of the procedures of Decker et al. (5). Twenty 150-mm plates of subconfluent CV-1 cells were infected with SV40 wt800 at 10 to 20 PFU per cell. At 36 to 38 h after infection, the culture medium was removed and then cells were washed twice with ice-cold TS buffer (20 mM Tris hydrochloride [pH 7.4], 137 mM NaCl, 5 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2) containing 0.25 M sucrose. All subsequent steps were carried out at 4°C. Cells were washed once with low-salt buffer (20 mM potassium HEPES [hydroxyethylpiperazineethanesulfonic acid] [pH 7.8], 5 mM potassium acetate, 0.5 mM MgCl2, 0.5 mM dithiothreitol), scraped off the plates, and then homogenized with five strokes of an all-glass Dounce homogenizer (pestle B; Wheaton Scientific). The resulting nuclei were pelleted by centrifugation (1,000 x g, 5 min) and suspended in 4 ml of high-salt buffer (20 mM potassium HEPES [pH 7.8], 500 mM potassium acetate, 0.5 mM MgCl2, 0.5 mM dithiothreitol). This nuclear suspension was then incubated on ice for 2 h with occasional mixing to extract SV40 viral chromosomes. After the nuclei were sedimented by centrifugation at 1,000 x g for 5 min and then at 20,000 x g for 15 min, the supernatant fraction was further centrifuged at 200,000 x g for 1 h (Hitachi RP 55S rotor), and the pelleted viral chromosomes were suspended in 200 ,il of low-salt buffer. Insoluble materials were removed by centrifugation at 10,000 x g for 10 min, and then the supernatant was stored at -80°C in aliquots. One microliter of this SV40 chromosome fraction contained 50 to 100 ng of SV40 DNA. To prepare 3H-labeled SV40 chromosomes, the culture medium was replaced 36 h after infection by 1 ml of Eagle minimal essential medium (Flow Laboratories, Inc.) per dish supplemented with 10% (vol/vol) dialyzed fetal calf serum, nonessential amino acids (Flow Laboratories), and 50 ,uCi of [methyl-3H]thymidine (50 Ci/mmol; ICN Biomedicals Inc.) per ml. After further incubation at 37°C for 2 h, viral chromosomes were extracted from the cells as described above, and this fraction was designated the 3H-SV40 chromosome fraction. To prepare SV40 [3H]DNA, the 3H-SV40 chromosome fraction was treated at 37°C for 1 h with 50 pLg of proteinase K (Boehringer Mannheim) per ml in the presence of 0.5% (wt/vol) sodium dodecyl sulfate, and then viral DNA was purified by phenol-chloroform extraction followed by ethanol precipitation. The specific radioactivity of this viral [3H]DNA was approximately 2 x 105 cpm/,ug of DNA. DNA replication reactions. The standard replication reaction mixture (50 [lI) contained the following substances: 30 mM Tris hydrochloride (pH 8.5); 3 mM MgCl2; 0.5 mM dithiothreitol; 2 mM ATP; CTP, GTP, and UTP (200 ,uM each); dATP, dCTP, and dGTP (100 ,uM each); 25 ,uM [methyl-3H]dTTP (1,500 cpm/pmol), 40 mM creatine phosphate (di-Tris salt; Sigma Chemical Co.); 20 ,ug of phosphocreatine kinase (Boehringer Mannheim) per ml; SV40 chromosomes (300 ng of SV40 DNA); HeLa cell extract (100 to
SV40 CHROMOSOME REPLICATION
4821
150 ,ug of protein); and 0.5 to 1 ,ug of purified SV40 T antigen. Where indicated, SV40 form I DNA, plasmid pBE102 DNA, FM3A cell extract, and/or polyomavirus large T antigen were substituted. Incubation was performed at 37°C, and the acid-insoluble radioactivity was measured. When the products of replication reactions were analyzed, 20 ,uM [a-32p] dCTP was used instead of [3H]dTTP and the concentration of cold dTTP was adjusted to 100 ,M. For density labeling of DNA products, 100 ,uM 5-bromo-dUTP (Sigma) was substituted for dTTP. Reactions were terminated by the addition of an equal volume of 10 mM EDTA and 1% (wt/vol) sodium dodecyl sulfate, and the mixture was then digested at 37°C for 1 h with 100 ,ug of proteinase K per ml. After phenolchloroform extraction, DNA containing 32 P-labeled products was recovered by ethanol precipitation in the presence of 30 p,g of yeast tRNA. Tritiated dTTP (40 Ci/mmol) was obtained from ICN Biomedicals Inc., and [o-32P]dCTP (3,000 Ci/mmol) was from Du Pont, NEN Research Products. Nonradioactive nucleoside triphosphates were purchased from Yamasa Shoyu Co., Ltd. Isopycnic centrifugation analysis. Replication reactions were performed in the presence of [a-32P]dCTP and 5-bromo-dUTP, and then the purified DNA products were digested with restriction endonucleases. After the digested DNA was fractionated by agarose gel electrophoresis, the indicated DNA fragments were isolated from the gel with DEAE-cellulose paper (DE81; Whatman, Inc.). These 32plabeled DNA fragments were mixed with EcoRI-digested SV40 [3H]DNA as an internal density marker (unsubstituted), and the mixture was adjusted to 58% (wt/wt) CsCI-10 mM Tris hydrochloride (pH 8.0)-i mM EDTA. A 3-ml portion of each sample was put into a 5-ml polyalomer ultracentrifuge tube and then overlaid with paraffin oil to be centrifuged at 85,000 x g for 70 h at 25°C (Hitachi RPS 50-2 rotor). Five-drop fractions were collected from the bottom of each tube onto Whatman 3MM filter paper (2.4 cm in diameter). The filters were then washed with 5% (wt/vol) trichloroacetic acid, washed twice with ethanol, and then dried. The 3H and 32P radioactivity was determined by a liquid scintillation system (Beckman Instruments, Inc.). For measurement of density gradients, blank samples were centrifuged in parallel, and 50-pl portions of the consecutive fractions were weighed in duplicate in tared capillary pipettes. Micrococcal nuclease digestion of replication products. To analyze digestion products by agarose gel electrophoresis, replication reactions were carried out in the presence of [a-32P]dCTP, after which cold dCTP was added to the reaction mixtures to a final concentration of 2 mM (100-fold molar excess) to minimize further incorporation of the radiolabel. Ten-microliter portions of the mixtures were diluted with 40 pA of 10 mM Tris hydrochloride (pH 7.5)-1.25 mM CaCl2 and then digested at 37°C with 0.5 U of micrococcal nuclease (Boehringer Mannheim) for the indicated times. After the reactions were terminated by the addition of an equal volume of 5 mM EDTA-1% (wt/vol) sodium dodecyl sulfate, the mixtures were further treated with proteinase K at a final concentration of 100 pug/ml (37°C, 1 h). Digested DNA products were recovered by phenol-chloroform extraction and, following ethanol precipitation in the presence of 30 ,ug of yeast tRNA, were fractionated by 2% agarose gel electrophoresis and then visualized by autoradiography. When the sensitivities to micrococcal nuclease were estimated for 32P-labeled replication products, digestion reactions were performed as described above except that an appropriate amount of either 3H-SV40 chromosome or SV40
4822
SUGASAWA ET AL.
[3H]DNA was included as an internal standard. Acid-insoluble radioactivity was measured for 3H and 32p, respectively, and the degree of digestion was evaluated as a percentage of the acid-solubilized radioactivity. Preparation of HeLa core histones. Core histones were purified from HeLa cell nuclei, which were obtained as residual material from the preparation of cell extracts. The nuclei from 109 cells were extracted with 0.2 M NaCl, pelleted down, and suspended in 4 ml of hypotonic buffer (20 mM sodium HEPES [pH 7.5], 5 mM KCl, 1.5 mM MgCl2, 1 mM dithiothreitol). The suspension was adjusted to 0.2 M H2SO4 and incubated at 4°C for 30 min with occasional agitation. After insoluble materials were removed by centrifugation at 20,000 x g for 20 min, core histones were precipitated at 4°C for 30 min in the presence of 5% (wt/wt) perchloric acid and then recovered by centrifugation (20,000 x g, 15 min). The pelleted core histones were washed twice with acetone, dried, and dissolved in water to be dialyzed against water at 4°C. The dialysate was neutralized by the addition of Na3PO4, and then aliquots were stored at -200C. Other methods. Restriction endonuclease digestion and gel electrophoresis techniques were carried out according to the procedures of Maniatis et al. (26). To prepare radiolabeled DNA size markers, bacteriophage 4XX174 RF DNA-HaeIII digests (Toyobo Co., Ltd.) were labeled at the 5' end by dephosphorylation with bacterial alkaline phosphatase (Takara Shuzo Co., Ltd.), followed by reaction with T4 polynucleotide kinase (Takara Shuzo) and [.y-32P]ATP (ICN Biomedicals Inc.; 7,000 Ci/mmol). Protein concentrations were determined by using the Bio-Rad protein assay kit with bovine serum albumin as the standard. RESULTS Preparation of SV40 chromosomes and construction of the reaction system. SV40 chromosomes were used as the templates for DNA synthesis and were prepared from a nuclear extract of virus-infected CV-1 cells. This viral chromosome fraction was free of host cell DNA contamination, and more than 90% of the viral DNA was in the covalently closed superhelical circular form (form I), as judged from the results of agarose gel electrophoresis (data not shown). Organization of the viral DNA into nucleosomes was demonstrated by the finding that partial micrococcal nuclease digestion of the fraction and subsequent agarose gel electrophoresis generated several ladder-like DNA bands with a unit length of approximately 200 base pairs (bp) (data not shown). To exclude the possibility that the chromosome fraction was contaminated with nonnucleosomal viral DNA, SV40 chromosomes were labeled in vivo with [3H]thymidine and analyzed by sedimentation through sucrose density gradients. As shown in Fig. 1A, the 3H-labeled viral chromosomes sedimented as a single peak, and no [3H]DNA was observed near the position corresponding to naked SV40 DNA (about 20S). In the presence of this SV40 chromosome fraction and purified SV40 T antigen, the crude extract prepared from HeLa cells catalyzed a significant incorporation of deoxyribonucleotides into acid-insoluble materials. After an approximately 15-min lag period, incorporation occurred for 2 h in a roughly linear manner (approximately 50 pmol of deoxyribonucleoside monophosphate [dNMP] per h/,Lg of DNA), and then a decline in the rate of DNA synthesis was observed (Fig. 2A). DNA synthesis in this system was completely dependent on the presence of the viral chromosomes (Fig. 2B) and the HeLa cell extract (Fig. 2C). Incor-
J. VIROL.
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FIG. 1. Sucrose gradient sedimentation analysis of SV40 chromosomes. Ten 150-mm dishes of CV-1 cells were infected with SV40, and then one-fifth of the cells were labeled with [3H]thymidine as described in Materials and Methods. A high-salt nuclear extract was prepared from the infected cells and loaded onto 12 ml of 5 to 40% (wt/vol) sucrose gradient before (0) and after (0) deproteinization. After centrifugation at 200,000 x g (Hitachi RPS 40T rotor) for 2.5 h at 4°C, 0.4-ml fractions were consecutively collected from the tops of the tubes. (A) Fifty microliters of each fraction was spotted onto a 2.4-cm disk of Whatman 3MM paper, and the 3H radioactivity of each fraction was then measured by liquid scintillation. (B) Ten microliters of each fraction derived from native chromosomes (0) was incubated at 37°C for 2 h with HeLa cell extract and SV40 T antigen to allow DNA products to be labeled with 32p. The products were fractionated by agarose gel electrophoresis and detected by autoradiography. The numbers of the fractions used for the reactions are indicated above each lane.
poration was also largely dependent on the addition of purified SV40 T antigen (Fig. 2D), but a low level of DNA synthesis occurred even in the absence of added T antigen (1.5 pmol of dNMP per h/,ug of DNA). It is likely that this T antigen-independent incorporation reflected DNA synthesis by a repair-type reaction and/or chain-elongation reaction, which occurred in replicating intermediate molecules of the viral chromosomes for which DNA replication had been initiated in vivo and was interrupted when the chromosomes were extracted. As shown in Table 1, the DNA synthesis required Mg2' and an ATP-regenerating system. Optimal concentrations of ATP and Mg2+ in this system were 2 mM and 3 mM, respectively. Incorporation was reduced by 50% under the conditions described for the various cell-free replication systems for purified SV40 ori-containing DNA (3 to 4 mM ATP and 7 to 8 mM MgCl2) (24, 44, 53). Purified SV40 form I DNA could also be replicated in our reaction system, and five- to sixfold higher incorporation was rou-
VOL. 64, 1990
SV40 CHROMOSOME REPLICATION
(A)
4823
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FIG. 2. Time course of SV40 chromosome replication and titration of the reaction components. (A) Reaction mixtures (50 ,ul) with (0) or without (0) SV40 T antigen incubated at 37°C for various times. (B and C) Reaction mixtures (50 ,ul) containing the indicated amounts of SV40 chromosomes (B) or crude extract from HeLa cells (C) incubated at 37°C for 2 h in the presence (@) or absence (0) of T antigen. (D) SV40 chromosomes incubated at 37°C for 2 h in the presence of various amounts of T antigen (50-,ul reactions). For each reaction, incorporation of [3H]dTTP into DNA products was assayed by determining the acid-insoluble radioactivity.
tinely obtained with it than with the chromosomal DNA template (Table 1). Analysis of synthesized DNA products. DNA products formed in the reactions described above were labeled with 32P and analyzed by agarose gel electrophoresis. As shown
Tag incubation time (min)
±Tag
120 15
30 60 120
TABLE 1. Requirements for DNA synthesis in SV40 chromosomesa Component(s) omitted
None (complete)
MgCl2 ATP
CTP, GTP, UTP dATP, dCTP, dGTP Creatine phosphate and phosphocreatine kinase HeLa cell extract SV40 large T antigen SV40 chromosomes SV40 chromosomes (+ SV40 form I DNA [300 ng])b
dNMP incorporated (pmol)
% of control incorporation
29.6 0.1 12.1 21.8 16.4 0.8
100 0.3 40.9 73.6 55.4 2.7
0.1 0.9 0.2 141
0.3 3.0 0.7 475
a Indicated components were removed from the standard reaction mixture, and the modified reaction mixtures (50 ,ul) were incubated at 37C for 2 h. The incorporation of [3H]dTTP into DNA products was assayed by determining the acid-insoluble radioactivity. b SV40 form I DNA (300 ng) was added to the standard reaction mixture in place of SV40 chromosomes.
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FIG. 3. Analysis of DNA products by agarose gel electrophoresis. As indicated above each lane, reaction mixtures containing 20 puM [a-32P]dCTP (10 p.Ci per reaction) were incubated for various times in the presence (lanes b through e) or absence (lane a) of T antigen. Purified DNA products were fractionated by 0.8% agarose gel electrophoresis and then detected by autoradiography. The positions of SV40 form I DNA (superhelical circular duplex), form II DNA (nicked circular duplex), and form III DNA (linear duplex) are indicated.
4824
J. VIROL.
SUGASAWA ET AL.
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Vol. 64, No. 10
0022-538X/90/104820-10$02.00/0 Copyright © 1990, American Society for Microbiology
Assembly of Nascent DNA into Nucleosome Structures in Simian Virus 40 Chromosomes by HeLa Cell Extract KAORU SUGASAWA,12t* YASUFUMI MURAKAMI,3 NORIMASA MIYAMOTO,3 FUMIO HANAOKA,"12 AND MICHIO UI1 Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113,1 Radiation Biology Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-01,2 and Tsukuba Life Science Research Center, RIKEN, Tsukuba, Ibaraki 305,3 Japan Received 2 February 1990/Accepted 9 July 1990
A soluble system was developed that could support DNA replication in simian virus 40 (SV40) chromosomes. DNA synthesis in this system required the presence of purified SV40 large tumor antigen, SV40 chromosomes prepared from virus-infected monkey cells, a crude extract from HeLa cells, and several low-molecular-weight components. In comparison to the replication of purified SV40 form I DNA, the rate of DNA synthesis was 15 to 20% in this system. DNA synthesis started near the replication origin of SV40 and proceeded bidirectionally in a semiconservative manner. Micrococcal nuclease digestion experiments revealed that the replicated DNA produced in this system became organized into a regularly spaced array of nucleosome core particles when an appropriate amount of purified HeLa core histones was added to the reaction mixture. SV40 form I DNA replicating under the same conditions was also assembled into nucleosomes, which were arranged in a rather dispersed manner and formed an aberrant chromatin structure.
In eucaryotic cells, the protein structure of chromosomes as well as DNA must be duplicated during the S phase of the cell division cycle. A number of observations have suggested that gene expression may somehow be regulated by the higher-order chromatin structure of each gene (reviewed in references 31 and 33). Although it is still uncertain whether chromatin structure actually plays any intrinsic role in gene regulation, it is possible that the events of DNA metabolism other than transcription, such as replication, recombination, and repair, may also be affected by the chromatin structure. Moreover, it has been suggested that some features of the chromatin structure, which influence the transcriptional activity of genes, may be stably propagated during cell proliferation to transmit a pattern of gene expression from parent to progeny cells (3, 8, 51, 52). Despite many elaborately designed experiments, however, the mechanisms involved in the propagation of chromatin structure in vivo remain poorly understood. For instance, even the modes of segregation and duplication of the most basic protein structure, the histone octamer, remain controversial. This is possibly because of the heterogeneity and intractability of the chromatin structure in intact cells (1, 4, 7, 12-16, 20-22, 32, 35-37, 39, 41, 45,
48). Under these circumstances, it was thought to be useful to develop a simple cell-free system, in which not only DNA but also its chromatin structure could be duplicated. Several cell-free systems have been described that can support faithful initiation and elongation in the replication of simian virus 40 (SV40) DNA or plasmid DNA containing SV40 ori. These systems used cell extracts from monkey cells (24) or human cells (25, 44, 53) fortified with separately purified SV40 T antigen as the source of replication proteins. Extensive biochemical and genetic analyses of these systems are underway to elucidate the molecular mechanisms involved
in the replication of viral DNA per se (reviewed in reference 17). More recently, Ishimi et al. (10) reported the reconstitution of the replication reactions of SV40 ori-containing plasmid DNA by using only purified enzymes and factors from HeLa cells plus the viral T antigen. However, the role of the chromatin structure has been little considered in these studies. Stillman (43) reported that the selective assembly of replicating SV40 DNA into a chromatin structure could occur with the addition to his reaction system of a nuclear extract from human 293 cells. More recently, Smith and Stillman (42) have reported the purification and characterization of a cellular factor required for replication-dependent chromatin assembly (CAF-I), although the template DNA used in their studies was purified to be nonnucleosomal. In addition, Decker et al. (5) reported a soluble system containing cytosol and nuclear extract fractions prepared from SV40-infected cells, which could initiate DNA replication in endogenous SV40 chromosomes, but the chromatin assembly of the newly synthesized DNA was not examined by these workers. In this paper, we describe the construction of a new soluble system that can support the DNA replication of SV40 chromosomes. This system contains SV40 chromosomes prepared from virus-infected CV-1 cells, a crude extract from uninfected HeLa cells, immunoaffinity-purified SV40 T antigen, and several other materials. The mode of viral DNA replication in this system mimicked that seen in vivo with respect to dependence on T antigen, bidirectional and semiconservative synthesis, and several other factors. Furthermore, digestion experiments with micrococcal nuclease strongly suggested that the newly synthesized DNA was assembled into a regularly spaced array of nucleosomes and that duplication of the nucleosome core structure must therefore occur in this reaction system.
Corresponding author. t Present address: Radiation Biology Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-01, Japan.
MATERIALS AND METHODS Extracts, T antigens, and DNA. Crude extracts from HeLa cells grown in suspension and SV40 large T antigen were prepared according to the procedures of Wobbe et al. (53).
*
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VOL. 64, 1990
Extracts from mouse mammary carcinoma FM3A cells were prepared in the same way as the extracts from HeLa cells. Polyomavirus large T antigen was produced in an insect cell line, SF27 cells infected with a recombinant baculovirus, vEV51LT (34). It was then purified by immunoaffinity chromatography by using a monoclonal antibody against polyomavirus large T antigen, F4 (30), as described by Murakami et al. (27). The construction of plasmid pBE102, which contains the replication origin of polyomavirus, has been described by Kern et al. (18). SV40 form I DNA was purchased from Bethesda Research Laboratories, Inc. Preparation of SV40 chromosomes. The SV40 chromosomal fraction was prepared on the basis of the procedures of Decker et al. (5). Twenty 150-mm plates of subconfluent CV-1 cells were infected with SV40 wt800 at 10 to 20 PFU per cell. At 36 to 38 h after infection, the culture medium was removed and then cells were washed twice with ice-cold TS buffer (20 mM Tris hydrochloride [pH 7.4], 137 mM NaCl, 5 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2) containing 0.25 M sucrose. All subsequent steps were carried out at 4°C. Cells were washed once with low-salt buffer (20 mM potassium HEPES [hydroxyethylpiperazineethanesulfonic acid] [pH 7.8], 5 mM potassium acetate, 0.5 mM MgCl2, 0.5 mM dithiothreitol), scraped off the plates, and then homogenized with five strokes of an all-glass Dounce homogenizer (pestle B; Wheaton Scientific). The resulting nuclei were pelleted by centrifugation (1,000 x g, 5 min) and suspended in 4 ml of high-salt buffer (20 mM potassium HEPES [pH 7.8], 500 mM potassium acetate, 0.5 mM MgCl2, 0.5 mM dithiothreitol). This nuclear suspension was then incubated on ice for 2 h with occasional mixing to extract SV40 viral chromosomes. After the nuclei were sedimented by centrifugation at 1,000 x g for 5 min and then at 20,000 x g for 15 min, the supernatant fraction was further centrifuged at 200,000 x g for 1 h (Hitachi RP 55S rotor), and the pelleted viral chromosomes were suspended in 200 ,il of low-salt buffer. Insoluble materials were removed by centrifugation at 10,000 x g for 10 min, and then the supernatant was stored at -80°C in aliquots. One microliter of this SV40 chromosome fraction contained 50 to 100 ng of SV40 DNA. To prepare 3H-labeled SV40 chromosomes, the culture medium was replaced 36 h after infection by 1 ml of Eagle minimal essential medium (Flow Laboratories, Inc.) per dish supplemented with 10% (vol/vol) dialyzed fetal calf serum, nonessential amino acids (Flow Laboratories), and 50 ,uCi of [methyl-3H]thymidine (50 Ci/mmol; ICN Biomedicals Inc.) per ml. After further incubation at 37°C for 2 h, viral chromosomes were extracted from the cells as described above, and this fraction was designated the 3H-SV40 chromosome fraction. To prepare SV40 [3H]DNA, the 3H-SV40 chromosome fraction was treated at 37°C for 1 h with 50 pLg of proteinase K (Boehringer Mannheim) per ml in the presence of 0.5% (wt/vol) sodium dodecyl sulfate, and then viral DNA was purified by phenol-chloroform extraction followed by ethanol precipitation. The specific radioactivity of this viral [3H]DNA was approximately 2 x 105 cpm/,ug of DNA. DNA replication reactions. The standard replication reaction mixture (50 [lI) contained the following substances: 30 mM Tris hydrochloride (pH 8.5); 3 mM MgCl2; 0.5 mM dithiothreitol; 2 mM ATP; CTP, GTP, and UTP (200 ,uM each); dATP, dCTP, and dGTP (100 ,uM each); 25 ,uM [methyl-3H]dTTP (1,500 cpm/pmol), 40 mM creatine phosphate (di-Tris salt; Sigma Chemical Co.); 20 ,ug of phosphocreatine kinase (Boehringer Mannheim) per ml; SV40 chromosomes (300 ng of SV40 DNA); HeLa cell extract (100 to
SV40 CHROMOSOME REPLICATION
4821
150 ,ug of protein); and 0.5 to 1 ,ug of purified SV40 T antigen. Where indicated, SV40 form I DNA, plasmid pBE102 DNA, FM3A cell extract, and/or polyomavirus large T antigen were substituted. Incubation was performed at 37°C, and the acid-insoluble radioactivity was measured. When the products of replication reactions were analyzed, 20 ,uM [a-32p] dCTP was used instead of [3H]dTTP and the concentration of cold dTTP was adjusted to 100 ,M. For density labeling of DNA products, 100 ,uM 5-bromo-dUTP (Sigma) was substituted for dTTP. Reactions were terminated by the addition of an equal volume of 10 mM EDTA and 1% (wt/vol) sodium dodecyl sulfate, and the mixture was then digested at 37°C for 1 h with 100 ,ug of proteinase K per ml. After phenolchloroform extraction, DNA containing 32 P-labeled products was recovered by ethanol precipitation in the presence of 30 p,g of yeast tRNA. Tritiated dTTP (40 Ci/mmol) was obtained from ICN Biomedicals Inc., and [o-32P]dCTP (3,000 Ci/mmol) was from Du Pont, NEN Research Products. Nonradioactive nucleoside triphosphates were purchased from Yamasa Shoyu Co., Ltd. Isopycnic centrifugation analysis. Replication reactions were performed in the presence of [a-32P]dCTP and 5-bromo-dUTP, and then the purified DNA products were digested with restriction endonucleases. After the digested DNA was fractionated by agarose gel electrophoresis, the indicated DNA fragments were isolated from the gel with DEAE-cellulose paper (DE81; Whatman, Inc.). These 32plabeled DNA fragments were mixed with EcoRI-digested SV40 [3H]DNA as an internal density marker (unsubstituted), and the mixture was adjusted to 58% (wt/wt) CsCI-10 mM Tris hydrochloride (pH 8.0)-i mM EDTA. A 3-ml portion of each sample was put into a 5-ml polyalomer ultracentrifuge tube and then overlaid with paraffin oil to be centrifuged at 85,000 x g for 70 h at 25°C (Hitachi RPS 50-2 rotor). Five-drop fractions were collected from the bottom of each tube onto Whatman 3MM filter paper (2.4 cm in diameter). The filters were then washed with 5% (wt/vol) trichloroacetic acid, washed twice with ethanol, and then dried. The 3H and 32P radioactivity was determined by a liquid scintillation system (Beckman Instruments, Inc.). For measurement of density gradients, blank samples were centrifuged in parallel, and 50-pl portions of the consecutive fractions were weighed in duplicate in tared capillary pipettes. Micrococcal nuclease digestion of replication products. To analyze digestion products by agarose gel electrophoresis, replication reactions were carried out in the presence of [a-32P]dCTP, after which cold dCTP was added to the reaction mixtures to a final concentration of 2 mM (100-fold molar excess) to minimize further incorporation of the radiolabel. Ten-microliter portions of the mixtures were diluted with 40 pA of 10 mM Tris hydrochloride (pH 7.5)-1.25 mM CaCl2 and then digested at 37°C with 0.5 U of micrococcal nuclease (Boehringer Mannheim) for the indicated times. After the reactions were terminated by the addition of an equal volume of 5 mM EDTA-1% (wt/vol) sodium dodecyl sulfate, the mixtures were further treated with proteinase K at a final concentration of 100 pug/ml (37°C, 1 h). Digested DNA products were recovered by phenol-chloroform extraction and, following ethanol precipitation in the presence of 30 ,ug of yeast tRNA, were fractionated by 2% agarose gel electrophoresis and then visualized by autoradiography. When the sensitivities to micrococcal nuclease were estimated for 32P-labeled replication products, digestion reactions were performed as described above except that an appropriate amount of either 3H-SV40 chromosome or SV40
4822
SUGASAWA ET AL.
[3H]DNA was included as an internal standard. Acid-insoluble radioactivity was measured for 3H and 32p, respectively, and the degree of digestion was evaluated as a percentage of the acid-solubilized radioactivity. Preparation of HeLa core histones. Core histones were purified from HeLa cell nuclei, which were obtained as residual material from the preparation of cell extracts. The nuclei from 109 cells were extracted with 0.2 M NaCl, pelleted down, and suspended in 4 ml of hypotonic buffer (20 mM sodium HEPES [pH 7.5], 5 mM KCl, 1.5 mM MgCl2, 1 mM dithiothreitol). The suspension was adjusted to 0.2 M H2SO4 and incubated at 4°C for 30 min with occasional agitation. After insoluble materials were removed by centrifugation at 20,000 x g for 20 min, core histones were precipitated at 4°C for 30 min in the presence of 5% (wt/wt) perchloric acid and then recovered by centrifugation (20,000 x g, 15 min). The pelleted core histones were washed twice with acetone, dried, and dissolved in water to be dialyzed against water at 4°C. The dialysate was neutralized by the addition of Na3PO4, and then aliquots were stored at -200C. Other methods. Restriction endonuclease digestion and gel electrophoresis techniques were carried out according to the procedures of Maniatis et al. (26). To prepare radiolabeled DNA size markers, bacteriophage 4XX174 RF DNA-HaeIII digests (Toyobo Co., Ltd.) were labeled at the 5' end by dephosphorylation with bacterial alkaline phosphatase (Takara Shuzo Co., Ltd.), followed by reaction with T4 polynucleotide kinase (Takara Shuzo) and [.y-32P]ATP (ICN Biomedicals Inc.; 7,000 Ci/mmol). Protein concentrations were determined by using the Bio-Rad protein assay kit with bovine serum albumin as the standard. RESULTS Preparation of SV40 chromosomes and construction of the reaction system. SV40 chromosomes were used as the templates for DNA synthesis and were prepared from a nuclear extract of virus-infected CV-1 cells. This viral chromosome fraction was free of host cell DNA contamination, and more than 90% of the viral DNA was in the covalently closed superhelical circular form (form I), as judged from the results of agarose gel electrophoresis (data not shown). Organization of the viral DNA into nucleosomes was demonstrated by the finding that partial micrococcal nuclease digestion of the fraction and subsequent agarose gel electrophoresis generated several ladder-like DNA bands with a unit length of approximately 200 base pairs (bp) (data not shown). To exclude the possibility that the chromosome fraction was contaminated with nonnucleosomal viral DNA, SV40 chromosomes were labeled in vivo with [3H]thymidine and analyzed by sedimentation through sucrose density gradients. As shown in Fig. 1A, the 3H-labeled viral chromosomes sedimented as a single peak, and no [3H]DNA was observed near the position corresponding to naked SV40 DNA (about 20S). In the presence of this SV40 chromosome fraction and purified SV40 T antigen, the crude extract prepared from HeLa cells catalyzed a significant incorporation of deoxyribonucleotides into acid-insoluble materials. After an approximately 15-min lag period, incorporation occurred for 2 h in a roughly linear manner (approximately 50 pmol of deoxyribonucleoside monophosphate [dNMP] per h/,Lg of DNA), and then a decline in the rate of DNA synthesis was observed (Fig. 2A). DNA synthesis in this system was completely dependent on the presence of the viral chromosomes (Fig. 2B) and the HeLa cell extract (Fig. 2C). Incor-
J. VIROL.
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FIG. 1. Sucrose gradient sedimentation analysis of SV40 chromosomes. Ten 150-mm dishes of CV-1 cells were infected with SV40, and then one-fifth of the cells were labeled with [3H]thymidine as described in Materials and Methods. A high-salt nuclear extract was prepared from the infected cells and loaded onto 12 ml of 5 to 40% (wt/vol) sucrose gradient before (0) and after (0) deproteinization. After centrifugation at 200,000 x g (Hitachi RPS 40T rotor) for 2.5 h at 4°C, 0.4-ml fractions were consecutively collected from the tops of the tubes. (A) Fifty microliters of each fraction was spotted onto a 2.4-cm disk of Whatman 3MM paper, and the 3H radioactivity of each fraction was then measured by liquid scintillation. (B) Ten microliters of each fraction derived from native chromosomes (0) was incubated at 37°C for 2 h with HeLa cell extract and SV40 T antigen to allow DNA products to be labeled with 32p. The products were fractionated by agarose gel electrophoresis and detected by autoradiography. The numbers of the fractions used for the reactions are indicated above each lane.
poration was also largely dependent on the addition of purified SV40 T antigen (Fig. 2D), but a low level of DNA synthesis occurred even in the absence of added T antigen (1.5 pmol of dNMP per h/,ug of DNA). It is likely that this T antigen-independent incorporation reflected DNA synthesis by a repair-type reaction and/or chain-elongation reaction, which occurred in replicating intermediate molecules of the viral chromosomes for which DNA replication had been initiated in vivo and was interrupted when the chromosomes were extracted. As shown in Table 1, the DNA synthesis required Mg2' and an ATP-regenerating system. Optimal concentrations of ATP and Mg2+ in this system were 2 mM and 3 mM, respectively. Incorporation was reduced by 50% under the conditions described for the various cell-free replication systems for purified SV40 ori-containing DNA (3 to 4 mM ATP and 7 to 8 mM MgCl2) (24, 44, 53). Purified SV40 form I DNA could also be replicated in our reaction system, and five- to sixfold higher incorporation was rou-
VOL. 64, 1990
SV40 CHROMOSOME REPLICATION
(A)
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50 100 150 200 HeLa cell extract (gg of protein)
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FIG. 2. Time course of SV40 chromosome replication and titration of the reaction components. (A) Reaction mixtures (50 ,ul) with (0) or without (0) SV40 T antigen incubated at 37°C for various times. (B and C) Reaction mixtures (50 ,ul) containing the indicated amounts of SV40 chromosomes (B) or crude extract from HeLa cells (C) incubated at 37°C for 2 h in the presence (@) or absence (0) of T antigen. (D) SV40 chromosomes incubated at 37°C for 2 h in the presence of various amounts of T antigen (50-,ul reactions). For each reaction, incorporation of [3H]dTTP into DNA products was assayed by determining the acid-insoluble radioactivity.
tinely obtained with it than with the chromosomal DNA template (Table 1). Analysis of synthesized DNA products. DNA products formed in the reactions described above were labeled with 32P and analyzed by agarose gel electrophoresis. As shown
Tag incubation time (min)
±Tag
120 15
30 60 120
TABLE 1. Requirements for DNA synthesis in SV40 chromosomesa Component(s) omitted
None (complete)
MgCl2 ATP
CTP, GTP, UTP dATP, dCTP, dGTP Creatine phosphate and phosphocreatine kinase HeLa cell extract SV40 large T antigen SV40 chromosomes SV40 chromosomes (+ SV40 form I DNA [300 ng])b
dNMP incorporated (pmol)
% of control incorporation
29.6 0.1 12.1 21.8 16.4 0.8
100 0.3 40.9 73.6 55.4 2.7
0.1 0.9 0.2 141
0.3 3.0 0.7 475
a Indicated components were removed from the standard reaction mixture, and the modified reaction mixtures (50 ,ul) were incubated at 37C for 2 h. The incorporation of [3H]dTTP into DNA products was assayed by determining the acid-insoluble radioactivity. b SV40 form I DNA (300 ng) was added to the standard reaction mixture in place of SV40 chromosomes.
SV40 (II) SV40 (111)-
-
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-
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a
b
c
d
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FIG. 3. Analysis of DNA products by agarose gel electrophoresis. As indicated above each lane, reaction mixtures containing 20 puM [a-32P]dCTP (10 p.Ci per reaction) were incubated for various times in the presence (lanes b through e) or absence (lane a) of T antigen. Purified DNA products were fractionated by 0.8% agarose gel electrophoresis and then detected by autoradiography. The positions of SV40 form I DNA (superhelical circular duplex), form II DNA (nicked circular duplex), and form III DNA (linear duplex) are indicated.
4824
J. VIROL.
SUGASAWA ET AL.
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time (min)
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