Biochhnica et Biophysica Acta, 1090 (1991) 305-310 © 1991 Elsevier Science Publishers B.V. All rights reserved 0167-4781/91/$03.50
305
BBAEXP 92304
Binding of simian virus 40 large tumor antigen to phospholipid vesicles Hiroshi Hirai, Noboru Takahashi, Shunji Natori and Kazuhisa Sekimizu Faculty of Pharmaceutical Sciences, Unicersity of Tokyo. Tokyo (Japan) (Received 2 January 1991)
Key words: SV40 T antigen; Phospholipid: Replication: Transcription
SV40 T antigen is the initiator protein of SV40 DNA replication. We examined the interaction of purified SV40 T antigen with phospholipids by (i) centrifugation analysis with phospholipid vesicles, (ii) filter binding assay and footprint analysis of T antigen binding to the replication origin of SV40 DNA and (iii) analysis of the initiation of SV40 DNA replication in vitro. In all cases, cardiolipin showed affinity for T antigen and inhibited its DNA binding capacity. Phosphatidylglycerol with unsaturated fatty acids also inhibited the binding of T antigen to the replication origin of SV40 DNA, whereas phosphatidylglycerol with saturated fatty acids did not. This finding suggested the importance of unsaturated fatty acids for the interaction of T antigen with phospholipids. Other phospholipids including phosphatidyiserine, phosphatidylinositol and phosphatidylethanolamine showed little or no affinity for T antigen.
Introduction DNA replication in cells has been proposed to be initiated on membranes [l]. In fact, membrane fractions of bacterial cells have been found to be enriched in replication origins [2-5]. Moreover, in Escherichia coli cells periodic association and dissociation of the replication origin (oriC) and membranes have been demonstrated [6]. These findings suggest that proteins that contribute to the initiation of DNA replication assemble on membranes to form an initiation complex of DNA replication. The DnaA protein plays a key role in the initiation of DNA replication in E. coli cells [7]. This protein binds specifically to the replication origin (oriC), resulting in the entrance of other replication proteins into the initiation complex [8]. Tightly bound adenine nucleotide regulates the activity of the DnaA protein: the ATP-bound form is active, and the ADP-bound form is inactive [9]. Cardiolipin and phosphatidylglycerol are candidates for reactivating the ADP-bound form of DnaA protein in cells [10]. These acidic phospholipids markedly reduce the affinity of the DnaA protein for ADP, and as a result, the DnaA protein is
Correspondence: K. Sekimizu, Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan.
converted to the ATP-bound form in the presence of a high concentration of ATP. On the other hand, excess concentrations of these phospholipids strongly inhibit the replication reaction of oriC plasmid in vitro because of their affinities for a DNA binding site on DnaA protein. These findings strongly suggest that phospholipids regulate the initiation of DNA replication in E. coli cells. There are also a number of reports suggesting the importance of phospholipids in the process of DNA (or RNA in the case of RNA virus) replication in eukaryotic cells. Newport found that nuclear membrane fractions were necessary for DNA synthesis in reconstituted nuclei [ll]. Eukaryotic DNA polymerases were shown to have affinity for phospholipids [12,13]. Moreover, recently phospholipids were reported to regulate poliovirus RNA replication [14]. At present, little is known about the initiator proteins of chromosomal DNA replication in eukaryotic cells. SV40 large tumor (T) antigen functions as an initiator protein of SV40 DNA replication in mammalian cells. Biochemical studies on a reconstituted system for SV40 DNA replication revealed that T antigen shares many characters with the DnaA protein; it binds specifically to the replication origin of SV40 DNA, resulting in formation of the initiation complex of DNA replication [15-18]. In this work, we examined the binding of SV40 T antigen to phospholipids. Re-
306 suits showed that, like DnaA protein, T antigen has an affinity for cardiolipin and phosphatidylglycerol.
Materials and Methods
Materials Cardiolipin (from bovine heart), phosphatidylglycerol (prepared from egg lecithin by treatment with phospholipase D in the presence of glycerol), phosphatidylinositol (from wheat germ or bovine liver), phosphatidylserine (from bovine spinal cord), phosphatidylcholine (from egg), L-a-phosphatidyl-vL-glycerol distearoyl and L-a-phosphatidyl-DL-glycerol dilinoleoyl were purchased from Lipid Products or from Avanti. PhosphoUpids were dried on the bottom of glass tubes and liposomes were prepared by vigorous vortex mixing with water. [a-a2p]dCTP and [a-32p]GTP were purchased from Amersham. DNA polymerase I large fragment (Klenow) was purchased from Takara. SV40 T antigen was purified as described by Simahis et al. [19]. SF27 cells transfected with recombinant baculovirus vEV55SVT [20] or vAcT2 [21] were lysed in buffer containing 1% NP-40. T antigen was affinity purified by monoclonal antibody PAb419 column chromatography. The final fraction of purified T antigen was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and its purity was estimated to be greater than 80%. Analysis of binding of T antigen to phospholipid cesicles T antigen (0.3 /~g) and phospholipids were incubated for 15 rain at 4°C in 100/zl of reaction mixture containing 30 mM Hepes-NaOH (pH 7.5), 7 mM MgCl 2, 0.5 mM DTI', 4 mM ATP, 40 mM creatine phosphate, 2/zg of creatine kinase and 0.2 mg/ml of bovine serum albumin. The mixture was then centrifuged in a Beckman TL100.3 rotor at 50000 rpm for 30 rain, The resulting precipitate was solubilized in buffer containing 1% SDS and analyzed by SDS-PAGE. Gels were stained by Coomassie brilliant blue and the amount of T antigen was determined by densitometric scanning. •. . . . . Filter binding assay A DNA fragment containing the SV40 replication origin was excised from pSVori ÷ by digestion with EcoRI and its 3' end was radiolabelled (1.105 cpm/pmol) by a filling reaction with the DNA po!ymerase l large fragment (Klenow). Probe DNA (5000 ~ m ) was incubated with T antigen (0.15/~g), 0.3 ~g pSVori + and various amounts of phospholipids in 50 /~1 of reaction mixture containing 30 mM Hepes-NaOH (pH 7.5), 7 mM MgCl2, 0.5 mM DTI', 4 mM ATP, 40 mM creatine phosphate, 1 / t g creatine kinase and 0.2 mg/ml bovine serum albumin at 4 °C for 15 rain. The sample was filtered through a nitrocellulose membrane
filter presoaked in washing buffer (30 mM HepesNaOH (pH 7.5), 7 mM MgC! 2 and 0.5 mM DTT). The filter was then washed with 10 ml of ice-cold washing buffer and dried under an infrared lamp. Radioactivity retained on the filter was measured in a liquid scintillation counter [22].
Footprint analysis The EcoRI site of the 3' end of the EcoRI-Sphl fragment of pSVori + containing the SV40 replication origin was radiolabelled by the filling reaction with DNA polymerase I large fragment (Klenow). Probe DNA (6.7' 10 4 cpm, 80 fmol) was incubated with T antigen and phospholipids in 50 g! of reaction mixture containing 30 mM Hepes-NaOH (pH 7.5), 7 mM MgCl 2, 10.5 mM DTT, 4 mM ATP, 40 mM creatine phosphate, 1 #g creatine kinase and 0.2 mg/ml bovine serum albumin at 4°C for 15 rain. The sample was digested with 200 ng of DNase I (RNase free, Worthington) at 30 °C for 90 s. The reaction was terminated by addition of EDTA, and [32p]DNA was extracted with phenol and chloroform and was analyzed by 8 M urea-6% polyacrylamide gel electrophoresis followed by autoradiography. SV40 DNA replication in vitro The reaction was carried out by a modification of the method of Wobbe et al. [23]. For analysis of the effect of phospholipids on formation of the initiation complex of DNA replication, T antigen (0.6/~g) and phospholipids were mixed in 30/~l of reaction mixt,,re containing 30 mM Hepes-NaOH (pH 7.5), 7 mM MgC! e, 0.5 mM DTI', 4 mM ATP, 200/zM concentrations of CTP, GTP and UTP, 40 mM creatine phosphate, 1 /zg of creatine kinase and pSVori + (0.3/~g) [24]. The mixture was preincubated at 4 °C for 15 rain and then incubated with a HeLa whole cell extract (400 ~tg of protein) at 37 ° C for 30 rain. DNA synthesis was initiated by addition of 100 /zM concentrations of dATP, dGTP and d'VFP and 25 /~M [a-32p]dCTP (1000-3000 cpm/pmol) at 37 ° C. For analysis of the effects of phospholipids on the elongation step, phospholipids were added after addition of deoxyribonucleoside triphosphates. Results
Binding of SV40 T antigen to phospholipids vesicles Fig. 1 and Table I show the results on the binding of purified SV40 T antigen to various phospholipids. In this experiment, T antigen was incubated with phospholipid vesicles, the mixtures were centrifuged and the amounts of precipitated T antigen were measured. T antigen was efficiently precipitated with cardiolipin. Phosphatidylglycerol also precipitated some T antigen, but less than cardiolipin. Phosphatidylserine
307 1
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Fig. I. Binding of T antigen to phospholipid vesicles. T antigen (0.3 /zg, lane l) was incubated with phospholipids (2 btl~') at 4 ° C for 15 rain. Samples were centrifuged and the amount of T antigen in the precipitates was analyzed by SDS-PAGE (hines 2-6). Lane 2, precipitates without "F antigen, without phospholipids', lan,¢s 3-6, precipitate with T antigen; hme 3, without phospholipids', hme 4, with cardiolipin; hme 5, with phosphatidylglycerol; hme 6, wnth phosphatidylserine. The arrows show migration positions of T antigen, bavine serum albumin and bmmophenol blue (BPB).
did not precipitate T antigen at all under these conditions. As shown in Fig. i, bovine serum albumin added in reaction mixtures was not precipitated in a phospholipid-dependent manner. Thus, we concluded that precipitation of T antigen is not due to the random sequestration into phospholipid vesicles. These results suggest that T antigen binds preferentiaUy to cardiolipin and that its replication activity can be modified by this phospholipid. Therefore, we investigated the interaction of phospholipids with T antigen by several other methods as described below.
Influence of phospholipids on the binding of T antigen to the replication origin of SV40 DNA We examined the influence of phospholipids on the binding of T antigen to SV40 DNA containing the replication origin by DNA filter binding assay. As shown in Fig. 2, cardiolipin from bovine heart markedly inhibited the binding of T antigen to the replication origin, but none of the other phospholipids tested affected this DNA binding activity of T antigen under these conditions. TABLE !
Binding of T antigen to phospholipid resides The gel in Fig. i was densitometric scanned and the amount of T antigen was determined. Less than 30% of T antigen was precipitated without phospholipid (Fig. 1, lane 3). Percentage of T antigen precipitated in a phospholipid-dependent manner is presented. The data are single points. This experiment was repeated three times. Phospholipid
T antigen precipitated (%)
Cardiolipin Phosphatidylglycerol Phosphatidylserine
100 30 0
0
!
O
0.75 Phospholipids (~g/so~t)
1.5
Fig. 2. Influence of phospholipids on tile binding of T antigen to Ihe SV4(I replication origin determined by filter binding assay. T antigen ((I.15/zg) was incubated with wlrious amounts of phospholipid vesicles and radiolabelled DNA containing tile replication origin of SV4(] (0.1)5 pmol, 51100 cpm). Each sample was filtered through a nitrocellulose membrane and the amount of radioactivity remaining on II,e filler was counted. This experiment was repeated twice. The date are single points, e, cardiolipin; O phosphalidylglycerol; I:], phosphatidylserine; II, phosphatidylinosilol; z~, phosphatidylcholine.
We confirmed the inhibitory action of cardiolipin on the specific binding activity of T antigen to the replication origin of SV40 DNA by DNase I footprint analysis. As reported previously [22], T antigen gave a clear footprint on site l and site II located in the replication origin (Fig. 3, lane 2). Cardiolipin inhibited the binding of T antigen to these sites (lanes 3 to 5). The binding ,,,-~ T antigen to site II was more sensitive to cardiolipin than that to site i; namely, at lower concentrations of cardiolipin (l to 3/~g), T antigen bound only to site I. The increased sensitivity to DNase ! in the upper range of the gel caused by T antigen (lane 2) is probably due to a conformational change of DNA accompanied with the binding of T antigen to the replication origin. Cardiolipin decreased the amount of radioactivity in this area (lanes 4 and 5) corresponding with the inhibitory action of this phospholipid on the binding activity of T antigen to the replication origin of SV40 DNA. Other acidic phospholipids, sucit as phosphatidylserine, phosphatidylglyceroi and phosphatidylinositol, were not inhibitory. DnaA protein has been shown to have higher affinity for phosphatidylglycerol with unsaturated fatty acids than for that with saturated fatty acids [25]. Thus, we tested whether SV40 T antigen also binds to synthetic phosphatidylglycerol with unsaturated fatty acids. The results in Fig. 4 show that synthetic phosphatidylglycerol with iinoleic acid (18" 2) inhibited the binding of T antigen as strongly as cardiolipin did, whereas phosphatidylglyceroi with stearic acid (18'0) caused little inhibition. Thus, the binding of T antigen to phosphatidylglycerol, like that of DnaA protein, requires unsaturated fatty acids.
308
12345678
hzfluence of phospholipids on the initiation step of SV40 DNA replication Since cardiolipin inhibits the binding of T antigen to the replication origin, we thought that it should block the initiation step of SV40 DNA replication. This possibility was tested in an in vitro SV40 DNA replica-
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Fig. 4. Influences of phosphatidylglycerol with unsaturated and saturated fatty acids on the binding of T antigen to the SV40 replication origin. DNase ! footprint analysis was performed as described in Fig. 2. Lane I, without T antigen; lanes 2-8, with T antigen; lanes 3, 4 and 5, with 3, 10 and 30 /zg of t.-a-phosphatidyl-DL-glycerol distearoyl: lanes 6, 7 and 8, with 3, 10 and 30/,tg of I.-a-phosphatidylt~u.-glycerol dUinoleoyl. The regions of sites ! and ii in the SV40 replication origin are indicated by arrows.
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Fig, 3, Influence of phospholipids on the binding of T antigen to the SV40 replication origin determined by footprint analysis. T antigen (1.1 /~g)was incubated with phospholipids and radiolabelled DNA containing the replication origin of SV40 at 4 °C for 15 min. The ~ample was further incubated with 200 ng of DNase ! at 30 ° C for 90 s, and the reaction was terminated by the addition of EDTA. DNA was extracted with phenol and chloroform and analyzed by 8 M urea-6% polyacrylamide gel electrophoresis followed by autoradiography. Lane !, without T antigen; lanes 2-14, with T antigen; lanes ! and 2, without phospholipid; lanes 3, 4 and 5, with 1, 3 and 12/~g of cardiolipin; lanes 6, 7 and 8, with 1, 3 and 12 tLg of phosphatidylserine; lanes 9, 10 and 11, with i, 3 and 12 /~g of phosphatidylglycerol; lanes 12, 13 and 14, with I, 3 and 12 /~g of phosphatidylinositol. The regions of sites I and II in the SV40 replication origin are indicated by arrows.
tion system with a HeLa whole cell extract [24]. Cardiolipin and phosphatidyiglycerol were shown to inhibit T antigen-dependent DNA synthesis. These phospholipids did not change a pattern of product DNA when analyzed by alkali agarose gel electrophoresis (data not shown). Thus, the preferential inhibition of the synthesis of leading strand or lagging strand was not observed. Formation of an initiation complex of DNA replication was measured by a staged reaction described by Wobbe et al. [23]. T antigen was preincubated with phospholipid vesicles and the DNA synthesis reaction was initiated by adding deoxyribonucleoside triphosphates. Under these conditions, reinitiation of DNA replication does not take place within 15 min after the addition of deoxyribonucleoside triphosphates [23]. Results showed that cardiolipin strongly inhibited DNA synthesis when added before formation of the
309 50
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Incubation t i m e ( min ) Fig. 5. Effects of phospholipids on the initiation step (A) and the elongation step (B) of SV40 DNA replication in vitro. (A) T antigen (0.6 p,g) and phospholipids (10 /.tg) were incubated at 4 ° C for 15 min. Incubation was continued with a whole extract of HeLa cells at 37 °C for 3(1 rain to form the initiation complex of DNA replication. DNA synthesis was performed with deoxyribonucleoside triphosphates at 37 o C. The amount of DNA synthesized was determined by measuring radioactivity incorporated into the acid-insoluble fraction. in B, phospholipids were added after addition of deoxyribonucleoside triphosphates. This experiment was repeated twice. The data are single points. El, with T antigen akme; o, with T antigen and cardiolipin; e, with T antigen and phosphatidylglycerol; II, with T antigen and phosphatidylinositol; * , without T antigen.
initiation complex (Fig. 5A). Phosphatidylglycerol caused partial inhibition. But phosphatidylinositol had scarcely any effect. Strikingly, when cardiolipin was added after formation of the initiation complex, its inhibitory effect on DNA synthesis was not apparent for the first 15 min after its addition (Fig. 5B). Therefore, cardiolipin seems to have a specific effect on the initiation step of replication, not on the elongation step.
Our results indicate that the binding of T antigen to the replication origin of SV40 DNA is preferentially inhibited by cardiolipin and phosphatidyiglycerol. T antigen is known to have DNA-dependent ATPase and DNA helicase activity. These phospholipids are likely to inhibit this activity of T antigen. ATP did not affect the binding capacity of T antigen to phospholipids (data not shown). Two factors in the structure of phospholipids seem to be important for their interaction with SV40 T antigen. One is a common motif found in both acidic phospholipids and DNA [27]. In fact, monoclonal antibodies that bind to both DNA and cardiolipin or phosphatidylglycerol have been reported [28,29]. T antigen may recognize this motif. The other factor is the presence of unsaturated fatty acids in the phospholipids. We demonstrated that phosphatidylglycerol with unsaturated fatty "tcids has a higher affinity for T antigen than that with saturated fatty acids. DnaA protein also binds to phosphatidylglycerol with unsaturated fatty acids [25]. The fluidity of biological membranes increases with increase in the content of unsaturated fatty acids in phospholipids [30]. Thus, T antigen and the DnaA protein may have higher affinity for membranes with higher fluidity. Various factors affect the fluidity of biological membranes [31], so they may also affect the initiation process of DNA replication through the action of replication initiator proteins. Both T antigen and DnaA protein act not only at DNA replication, but also at transcription [32,33]. In fact, we showed that repression by T antigen of SV40 early gene transcription is relieved by cardiolipin (data not shown). We suggest here that phospholipids may also affect gene expression in cells through the interaction with other DNA binding proteins that regulate transcription. To date, little attention has been paid to the possibility that phospholipids participate in regulation of DNA replication and transcription. Further studies on the interaction of DNA binding proteins and phospholipids are necessary for understanding this new aspect of the biological importance of phospholipids in cell division and gone expression. Binding of SV40 T antigen to the plasma membrane of SV40 virus-infected and transformed cells was reported [34]. Our results obtained here could contribute to an understanding of this particular property of T antigen.
Discussion In this study, we showed that T antigen, the initiator protein of SV40 DNA replication, has a capacity to bind to phospholipids. DnaA protein, the initiator protein of E. coil chromosomal DNA replication, was previously shown to bind to phospholipids [10,26]. Thus, these two replication initiator proteins share the common character of phospholipid binding.
Acknowledgements We wish to thank Drs. F. Hanaoka, K. Sugasawa, Y. Murakami and Y. Ishimi for providing us with materials for construction of the SV40 in vitro replication system. We are also indebted to Drs. M.K. Bradley and L.K. Miller for gifts of recombinant baculoviruses.
310 References I Jacob, F., Brenner, S. and Cuzin, F. (1963) Cold Spring tlarbor Syrup. Quant. Biol. 28, 329-348. 2 Sueoka, N. and Quinn, W.G. (ltJt~8) Cold Spring Harbor Symp. Quant. Biol. 33, 695-705. 3 Yamaguchi, K. and Yoshikawa, tl. (1975)J. Bacteriol. 124, 10301033. 4 Nicolaidis, A.A. and Holhmd, I.B. (19781 J. Bacteriol. 135, 178189. 5 W.-Watz, tt. and Norqvist, A. (19781 J. Baeteriol. 140, 43-49. t~ Gayama, S., Kataoka, T., Wachi, M., Tamura, G. and Nagai, K. (1990) EMBO J. 9, 3761-3765. 7 Fuller, R.S., Funnell, B.E. and Kornberg, A. (19841 Cell 38, 889-~10, 8 Sekimizu, K., Bramhill, D. and Kornberg, A. (1~88) J. Biol. Chem. 263, 7124-7130. L) Sekimizu, K., Bramhill, D. and Kornberg, A. (1987) Cell 50, 259-265. l0 Sekimizu, K. and Kornberg, A, (IL,~88)J. Biol. Chem, 263, 71317135. I I Newport, J. (19871 Cell 48, 205-217. 12 Sylvi';, V., Curlin, G., Norman, J., Stec, J. and Busbce, D. (It~88) C~ll 54, 651-658, 13 Yoshida, S., T.-Koizumi, K, and Kojima, K, (IL~89) Biochim. Biophys. Acta 1007, 61-6f~, 14 Guinea, R. and Carrasco, L. (It~t}il) EMBO J. 9, 2011-2016. 15 "Ijian, R. (1978)Cell 13, 165-179. 16 Shortle, D,R., Margolskge, R.F. and Nathans, D. (1979) Proc. Natl. Acad. Sci. USA 76, 6128-6131. 17 lshimi, Y., Claude, A., Bullock, P. and tlurwitz, J. (19881 J. Biol. Chem. 263, 19723-19733. 18 Hay, R.T. and Russell, W.C. (!t189) Biochem. J. 258, 3-16.
19 Simanis, V. and Lane, D.P. (1985) Virology 144, 88-100. 20 O'Reilly, D.R. and Miller, L.K. (19881 J. Virol. 62, 3109-3119. 21 Murphy, C.I., Weiner, B., Bikel, !., P.-Worms, H., Bradley, M.K. and Livingston, D.M. (1988)J. Virol. 62, 2951-2959. 22 Borowiec, J.A. and Hurwitz, J. (1988) Proc. Natl. Acad. Sci. USA 85, 64-68. 23 Wobbe, C.R., Dean, F., Murakami, Y., Weissbach, L. and llurwilz, J. 11986) Pro¢. Natl. Acad. S~:i. USA 83, 4bi2-4bi(~. 24 Wobbe, C.R., Dean, F., Weissbach, L. and Hurwitz, J. (1985) Proc. Natl. Acad. Sci. USA 82, 5710-5714. 25 Yung, B.Y.-M. and Kornberg, A. (1988) Proc. Natl. Acad. Sci. USA 85, 7202-7205. 26 Sekimizu, K., Yung, B.Y.-M, and Kornberg, A. (19881 J. Biol. Chem. 263, 7136-7140. 27 lnoue, K,, Nojima, S. and Tomizawa, T. (1%51 J. Biochem. (Tokyo) 57, 824-826, 28 Lafer, E.M., Rauch, J., Andrzejewski, C., Mudd, D., Furie, B., Furie, B., Schwartz, R.S. and Stollar, B.D. (1981) J. Exp. Med. 153, 897-~0cj. 2~J Shoenfekl, Y., Raueh, J., Massicotte, H., Datta, S.K., A.-Schwartz, J., Stollar, B.D. and Schwartz, R.S. (1~,~83) N. E. J. Med. 3118, 414-420. 30 Finean, J.B., Coleman, R. and Michell, R.ll. (1~}84) in Membranes and Their Cellular Functions, 3rd Edn., Blackwell Scientific Publications, Oxford. 31 Tilcock, C.P.S. (19861 Chem. Phys, Lip. 40, 109-125. 32 Rio, D., Robbins, A., Myers, R. am! Tjian, R. (1980) Proc. Natl. Acad. Sci. USA 77, 5706-5710. 33 Myers, R.M., Rio, D.C., Robbins, A.K. and Tjian, R. (1981) Cell 25, 373-384. 34 Lange-Mutschlcr, J. and Henning, R. (1983) Virology 127, 333344.
305
BBAEXP 92304
Binding of simian virus 40 large tumor antigen to phospholipid vesicles Hiroshi Hirai, Noboru Takahashi, Shunji Natori and Kazuhisa Sekimizu Faculty of Pharmaceutical Sciences, Unicersity of Tokyo. Tokyo (Japan) (Received 2 January 1991)
Key words: SV40 T antigen; Phospholipid: Replication: Transcription
SV40 T antigen is the initiator protein of SV40 DNA replication. We examined the interaction of purified SV40 T antigen with phospholipids by (i) centrifugation analysis with phospholipid vesicles, (ii) filter binding assay and footprint analysis of T antigen binding to the replication origin of SV40 DNA and (iii) analysis of the initiation of SV40 DNA replication in vitro. In all cases, cardiolipin showed affinity for T antigen and inhibited its DNA binding capacity. Phosphatidylglycerol with unsaturated fatty acids also inhibited the binding of T antigen to the replication origin of SV40 DNA, whereas phosphatidylglycerol with saturated fatty acids did not. This finding suggested the importance of unsaturated fatty acids for the interaction of T antigen with phospholipids. Other phospholipids including phosphatidyiserine, phosphatidylinositol and phosphatidylethanolamine showed little or no affinity for T antigen.
Introduction DNA replication in cells has been proposed to be initiated on membranes [l]. In fact, membrane fractions of bacterial cells have been found to be enriched in replication origins [2-5]. Moreover, in Escherichia coli cells periodic association and dissociation of the replication origin (oriC) and membranes have been demonstrated [6]. These findings suggest that proteins that contribute to the initiation of DNA replication assemble on membranes to form an initiation complex of DNA replication. The DnaA protein plays a key role in the initiation of DNA replication in E. coli cells [7]. This protein binds specifically to the replication origin (oriC), resulting in the entrance of other replication proteins into the initiation complex [8]. Tightly bound adenine nucleotide regulates the activity of the DnaA protein: the ATP-bound form is active, and the ADP-bound form is inactive [9]. Cardiolipin and phosphatidylglycerol are candidates for reactivating the ADP-bound form of DnaA protein in cells [10]. These acidic phospholipids markedly reduce the affinity of the DnaA protein for ADP, and as a result, the DnaA protein is
Correspondence: K. Sekimizu, Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan.
converted to the ATP-bound form in the presence of a high concentration of ATP. On the other hand, excess concentrations of these phospholipids strongly inhibit the replication reaction of oriC plasmid in vitro because of their affinities for a DNA binding site on DnaA protein. These findings strongly suggest that phospholipids regulate the initiation of DNA replication in E. coli cells. There are also a number of reports suggesting the importance of phospholipids in the process of DNA (or RNA in the case of RNA virus) replication in eukaryotic cells. Newport found that nuclear membrane fractions were necessary for DNA synthesis in reconstituted nuclei [ll]. Eukaryotic DNA polymerases were shown to have affinity for phospholipids [12,13]. Moreover, recently phospholipids were reported to regulate poliovirus RNA replication [14]. At present, little is known about the initiator proteins of chromosomal DNA replication in eukaryotic cells. SV40 large tumor (T) antigen functions as an initiator protein of SV40 DNA replication in mammalian cells. Biochemical studies on a reconstituted system for SV40 DNA replication revealed that T antigen shares many characters with the DnaA protein; it binds specifically to the replication origin of SV40 DNA, resulting in formation of the initiation complex of DNA replication [15-18]. In this work, we examined the binding of SV40 T antigen to phospholipids. Re-
306 suits showed that, like DnaA protein, T antigen has an affinity for cardiolipin and phosphatidylglycerol.
Materials and Methods
Materials Cardiolipin (from bovine heart), phosphatidylglycerol (prepared from egg lecithin by treatment with phospholipase D in the presence of glycerol), phosphatidylinositol (from wheat germ or bovine liver), phosphatidylserine (from bovine spinal cord), phosphatidylcholine (from egg), L-a-phosphatidyl-vL-glycerol distearoyl and L-a-phosphatidyl-DL-glycerol dilinoleoyl were purchased from Lipid Products or from Avanti. PhosphoUpids were dried on the bottom of glass tubes and liposomes were prepared by vigorous vortex mixing with water. [a-a2p]dCTP and [a-32p]GTP were purchased from Amersham. DNA polymerase I large fragment (Klenow) was purchased from Takara. SV40 T antigen was purified as described by Simahis et al. [19]. SF27 cells transfected with recombinant baculovirus vEV55SVT [20] or vAcT2 [21] were lysed in buffer containing 1% NP-40. T antigen was affinity purified by monoclonal antibody PAb419 column chromatography. The final fraction of purified T antigen was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and its purity was estimated to be greater than 80%. Analysis of binding of T antigen to phospholipid cesicles T antigen (0.3 /~g) and phospholipids were incubated for 15 rain at 4°C in 100/zl of reaction mixture containing 30 mM Hepes-NaOH (pH 7.5), 7 mM MgCl 2, 0.5 mM DTI', 4 mM ATP, 40 mM creatine phosphate, 2/zg of creatine kinase and 0.2 mg/ml of bovine serum albumin. The mixture was then centrifuged in a Beckman TL100.3 rotor at 50000 rpm for 30 rain, The resulting precipitate was solubilized in buffer containing 1% SDS and analyzed by SDS-PAGE. Gels were stained by Coomassie brilliant blue and the amount of T antigen was determined by densitometric scanning. •. . . . . Filter binding assay A DNA fragment containing the SV40 replication origin was excised from pSVori ÷ by digestion with EcoRI and its 3' end was radiolabelled (1.105 cpm/pmol) by a filling reaction with the DNA po!ymerase l large fragment (Klenow). Probe DNA (5000 ~ m ) was incubated with T antigen (0.15/~g), 0.3 ~g pSVori + and various amounts of phospholipids in 50 /~1 of reaction mixture containing 30 mM Hepes-NaOH (pH 7.5), 7 mM MgCl2, 0.5 mM DTI', 4 mM ATP, 40 mM creatine phosphate, 1 / t g creatine kinase and 0.2 mg/ml bovine serum albumin at 4 °C for 15 rain. The sample was filtered through a nitrocellulose membrane
filter presoaked in washing buffer (30 mM HepesNaOH (pH 7.5), 7 mM MgC! 2 and 0.5 mM DTT). The filter was then washed with 10 ml of ice-cold washing buffer and dried under an infrared lamp. Radioactivity retained on the filter was measured in a liquid scintillation counter [22].
Footprint analysis The EcoRI site of the 3' end of the EcoRI-Sphl fragment of pSVori + containing the SV40 replication origin was radiolabelled by the filling reaction with DNA polymerase I large fragment (Klenow). Probe DNA (6.7' 10 4 cpm, 80 fmol) was incubated with T antigen and phospholipids in 50 g! of reaction mixture containing 30 mM Hepes-NaOH (pH 7.5), 7 mM MgCl 2, 10.5 mM DTT, 4 mM ATP, 40 mM creatine phosphate, 1 #g creatine kinase and 0.2 mg/ml bovine serum albumin at 4°C for 15 rain. The sample was digested with 200 ng of DNase I (RNase free, Worthington) at 30 °C for 90 s. The reaction was terminated by addition of EDTA, and [32p]DNA was extracted with phenol and chloroform and was analyzed by 8 M urea-6% polyacrylamide gel electrophoresis followed by autoradiography. SV40 DNA replication in vitro The reaction was carried out by a modification of the method of Wobbe et al. [23]. For analysis of the effect of phospholipids on formation of the initiation complex of DNA replication, T antigen (0.6/~g) and phospholipids were mixed in 30/~l of reaction mixt,,re containing 30 mM Hepes-NaOH (pH 7.5), 7 mM MgC! e, 0.5 mM DTI', 4 mM ATP, 200/zM concentrations of CTP, GTP and UTP, 40 mM creatine phosphate, 1 /zg of creatine kinase and pSVori + (0.3/~g) [24]. The mixture was preincubated at 4 °C for 15 rain and then incubated with a HeLa whole cell extract (400 ~tg of protein) at 37 ° C for 30 rain. DNA synthesis was initiated by addition of 100 /zM concentrations of dATP, dGTP and d'VFP and 25 /~M [a-32p]dCTP (1000-3000 cpm/pmol) at 37 ° C. For analysis of the effects of phospholipids on the elongation step, phospholipids were added after addition of deoxyribonucleoside triphosphates. Results
Binding of SV40 T antigen to phospholipids vesicles Fig. 1 and Table I show the results on the binding of purified SV40 T antigen to various phospholipids. In this experiment, T antigen was incubated with phospholipid vesicles, the mixtures were centrifuged and the amounts of precipitated T antigen were measured. T antigen was efficiently precipitated with cardiolipin. Phosphatidylglycerol also precipitated some T antigen, but less than cardiolipin. Phosphatidylserine
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Fig. I. Binding of T antigen to phospholipid vesicles. T antigen (0.3 /zg, lane l) was incubated with phospholipids (2 btl~') at 4 ° C for 15 rain. Samples were centrifuged and the amount of T antigen in the precipitates was analyzed by SDS-PAGE (hines 2-6). Lane 2, precipitates without "F antigen, without phospholipids', lan,¢s 3-6, precipitate with T antigen; hme 3, without phospholipids', hme 4, with cardiolipin; hme 5, with phosphatidylglycerol; hme 6, wnth phosphatidylserine. The arrows show migration positions of T antigen, bavine serum albumin and bmmophenol blue (BPB).
did not precipitate T antigen at all under these conditions. As shown in Fig. i, bovine serum albumin added in reaction mixtures was not precipitated in a phospholipid-dependent manner. Thus, we concluded that precipitation of T antigen is not due to the random sequestration into phospholipid vesicles. These results suggest that T antigen binds preferentiaUy to cardiolipin and that its replication activity can be modified by this phospholipid. Therefore, we investigated the interaction of phospholipids with T antigen by several other methods as described below.
Influence of phospholipids on the binding of T antigen to the replication origin of SV40 DNA We examined the influence of phospholipids on the binding of T antigen to SV40 DNA containing the replication origin by DNA filter binding assay. As shown in Fig. 2, cardiolipin from bovine heart markedly inhibited the binding of T antigen to the replication origin, but none of the other phospholipids tested affected this DNA binding activity of T antigen under these conditions. TABLE !
Binding of T antigen to phospholipid resides The gel in Fig. i was densitometric scanned and the amount of T antigen was determined. Less than 30% of T antigen was precipitated without phospholipid (Fig. 1, lane 3). Percentage of T antigen precipitated in a phospholipid-dependent manner is presented. The data are single points. This experiment was repeated three times. Phospholipid
T antigen precipitated (%)
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We confirmed the inhibitory action of cardiolipin on the specific binding activity of T antigen to the replication origin of SV40 DNA by DNase I footprint analysis. As reported previously [22], T antigen gave a clear footprint on site l and site II located in the replication origin (Fig. 3, lane 2). Cardiolipin inhibited the binding of T antigen to these sites (lanes 3 to 5). The binding ,,,-~ T antigen to site II was more sensitive to cardiolipin than that to site i; namely, at lower concentrations of cardiolipin (l to 3/~g), T antigen bound only to site I. The increased sensitivity to DNase ! in the upper range of the gel caused by T antigen (lane 2) is probably due to a conformational change of DNA accompanied with the binding of T antigen to the replication origin. Cardiolipin decreased the amount of radioactivity in this area (lanes 4 and 5) corresponding with the inhibitory action of this phospholipid on the binding activity of T antigen to the replication origin of SV40 DNA. Other acidic phospholipids, sucit as phosphatidylserine, phosphatidylglyceroi and phosphatidylinositol, were not inhibitory. DnaA protein has been shown to have higher affinity for phosphatidylglycerol with unsaturated fatty acids than for that with saturated fatty acids [25]. Thus, we tested whether SV40 T antigen also binds to synthetic phosphatidylglycerol with unsaturated fatty acids. The results in Fig. 4 show that synthetic phosphatidylglycerol with iinoleic acid (18" 2) inhibited the binding of T antigen as strongly as cardiolipin did, whereas phosphatidylglyceroi with stearic acid (18'0) caused little inhibition. Thus, the binding of T antigen to phosphatidylglycerol, like that of DnaA protein, requires unsaturated fatty acids.
308
12345678
hzfluence of phospholipids on the initiation step of SV40 DNA replication Since cardiolipin inhibits the binding of T antigen to the replication origin, we thought that it should block the initiation step of SV40 DNA replication. This possibility was tested in an in vitro SV40 DNA replica-
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Fig, 3, Influence of phospholipids on the binding of T antigen to the SV40 replication origin determined by footprint analysis. T antigen (1.1 /~g)was incubated with phospholipids and radiolabelled DNA containing the replication origin of SV40 at 4 °C for 15 min. The ~ample was further incubated with 200 ng of DNase ! at 30 ° C for 90 s, and the reaction was terminated by the addition of EDTA. DNA was extracted with phenol and chloroform and analyzed by 8 M urea-6% polyacrylamide gel electrophoresis followed by autoradiography. Lane !, without T antigen; lanes 2-14, with T antigen; lanes ! and 2, without phospholipid; lanes 3, 4 and 5, with 1, 3 and 12/~g of cardiolipin; lanes 6, 7 and 8, with 1, 3 and 12 tLg of phosphatidylserine; lanes 9, 10 and 11, with i, 3 and 12 /~g of phosphatidylglycerol; lanes 12, 13 and 14, with I, 3 and 12 /~g of phosphatidylinositol. The regions of sites I and II in the SV40 replication origin are indicated by arrows.
tion system with a HeLa whole cell extract [24]. Cardiolipin and phosphatidyiglycerol were shown to inhibit T antigen-dependent DNA synthesis. These phospholipids did not change a pattern of product DNA when analyzed by alkali agarose gel electrophoresis (data not shown). Thus, the preferential inhibition of the synthesis of leading strand or lagging strand was not observed. Formation of an initiation complex of DNA replication was measured by a staged reaction described by Wobbe et al. [23]. T antigen was preincubated with phospholipid vesicles and the DNA synthesis reaction was initiated by adding deoxyribonucleoside triphosphates. Under these conditions, reinitiation of DNA replication does not take place within 15 min after the addition of deoxyribonucleoside triphosphates [23]. Results showed that cardiolipin strongly inhibited DNA synthesis when added before formation of the
309 50
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Incubation t i m e ( min ) Fig. 5. Effects of phospholipids on the initiation step (A) and the elongation step (B) of SV40 DNA replication in vitro. (A) T antigen (0.6 p,g) and phospholipids (10 /.tg) were incubated at 4 ° C for 15 min. Incubation was continued with a whole extract of HeLa cells at 37 °C for 3(1 rain to form the initiation complex of DNA replication. DNA synthesis was performed with deoxyribonucleoside triphosphates at 37 o C. The amount of DNA synthesized was determined by measuring radioactivity incorporated into the acid-insoluble fraction. in B, phospholipids were added after addition of deoxyribonucleoside triphosphates. This experiment was repeated twice. The data are single points. El, with T antigen akme; o, with T antigen and cardiolipin; e, with T antigen and phosphatidylglycerol; II, with T antigen and phosphatidylinositol; * , without T antigen.
initiation complex (Fig. 5A). Phosphatidylglycerol caused partial inhibition. But phosphatidylinositol had scarcely any effect. Strikingly, when cardiolipin was added after formation of the initiation complex, its inhibitory effect on DNA synthesis was not apparent for the first 15 min after its addition (Fig. 5B). Therefore, cardiolipin seems to have a specific effect on the initiation step of replication, not on the elongation step.
Our results indicate that the binding of T antigen to the replication origin of SV40 DNA is preferentially inhibited by cardiolipin and phosphatidyiglycerol. T antigen is known to have DNA-dependent ATPase and DNA helicase activity. These phospholipids are likely to inhibit this activity of T antigen. ATP did not affect the binding capacity of T antigen to phospholipids (data not shown). Two factors in the structure of phospholipids seem to be important for their interaction with SV40 T antigen. One is a common motif found in both acidic phospholipids and DNA [27]. In fact, monoclonal antibodies that bind to both DNA and cardiolipin or phosphatidylglycerol have been reported [28,29]. T antigen may recognize this motif. The other factor is the presence of unsaturated fatty acids in the phospholipids. We demonstrated that phosphatidylglycerol with unsaturated fatty "tcids has a higher affinity for T antigen than that with saturated fatty acids. DnaA protein also binds to phosphatidylglycerol with unsaturated fatty acids [25]. The fluidity of biological membranes increases with increase in the content of unsaturated fatty acids in phospholipids [30]. Thus, T antigen and the DnaA protein may have higher affinity for membranes with higher fluidity. Various factors affect the fluidity of biological membranes [31], so they may also affect the initiation process of DNA replication through the action of replication initiator proteins. Both T antigen and DnaA protein act not only at DNA replication, but also at transcription [32,33]. In fact, we showed that repression by T antigen of SV40 early gene transcription is relieved by cardiolipin (data not shown). We suggest here that phospholipids may also affect gene expression in cells through the interaction with other DNA binding proteins that regulate transcription. To date, little attention has been paid to the possibility that phospholipids participate in regulation of DNA replication and transcription. Further studies on the interaction of DNA binding proteins and phospholipids are necessary for understanding this new aspect of the biological importance of phospholipids in cell division and gone expression. Binding of SV40 T antigen to the plasma membrane of SV40 virus-infected and transformed cells was reported [34]. Our results obtained here could contribute to an understanding of this particular property of T antigen.
Discussion In this study, we showed that T antigen, the initiator protein of SV40 DNA replication, has a capacity to bind to phospholipids. DnaA protein, the initiator protein of E. coil chromosomal DNA replication, was previously shown to bind to phospholipids [10,26]. Thus, these two replication initiator proteins share the common character of phospholipid binding.
Acknowledgements We wish to thank Drs. F. Hanaoka, K. Sugasawa, Y. Murakami and Y. Ishimi for providing us with materials for construction of the SV40 in vitro replication system. We are also indebted to Drs. M.K. Bradley and L.K. Miller for gifts of recombinant baculoviruses.
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