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Topoisomerase activity associated with polyoma virus large tumor antigen
ELSEVIER
B iochimica et Biophysica Acta 1262 (1995) 59-63
Biochim~ic~a et BiophysicaA~ta
Topoisomerase activity associated with polyoma virus large tumor antigen Attila Marton, Bernadette Marko, Louis Delbecchi, Pierre Bourgaux
*
Department of Microbiology, Faculty of Medicine, Universit~ de Sherbrooke, 3001, 12e Avenue Nord, Sherbrooke, Quebec, J1H 5N4, Canada Received 24 November 1994; accepted 22 February 1995
Abstract
Polyomavirus (Py) large tumor antigen (LT) was produced in mammalian or insect cells infected with a suitable viral expression vector, and purified by a procedure combining immunoprecipitation with ion-exchange chromatography. Fractions containing the bulk of LT displayed a DNA-relaxing activity (LT-topo) which could be attributed neither to topoisomerase II (topo II) nor to topoisomerase I (topo I) encoded by the cell or the viral vector. On the one hand, LT-topo relaxed pBR322 DNA in a reaction which, unlike that characteristic of topo II, was ATP-independent and inhibited by camptothecin. On the other hand, serum from scleroderma patients which strongly inhibited calf thymus topo I had no effect on LT-topo, which absolutely required Mg 2+ ions to relax DNA. Thus, LT-topo is either inherent to LT or belongs to a LT-bound enzyme similar to, but distinct from, topo I. Keywords: Polyoma large T antigen; DNA relaxation; Camptothecin; Scleroderma; Magnesium ion-dependent topoisomerase
1. Introduction
2. Materials and methods
The large tumor antigens (LTs) of SV40 and polyoma virus (Py) are multifunctional proteins known to interact specifically with DNA as well as with cellular proteins [1]. Recently, we and others [2,3] have shown that purified preparations of SV40 LT have topoisomerase activity, a finding of potential significance with respect to the role of LT in replication [1], transcription [4] and recombination [5,6]. Available data do not allow us to decide whether this activity was inherent to or associated with SV40 LT, even though trivial contamination could be clearly excluded [2,3]. Py LT is structurally and functionally similar but not identical to SV40 LT. For example, SV40 LT carries a transforming function [7,8] and a function allowing the complementation of adenovirus [9], both of which are absent from Py LT [10]. Also, SV40 LT, but not Py LT, interacts with the host protein p53 [11,12]. It was thus conceivable that purified Py LT would lack the DNA-relaxing activity associated with SV40 LT. We show here that preparations of Py LT display a DNA-relaxing activity (LT-topo) similar to that of SV40 LT [2,3], but clearly distinct from those of potential contaminants such as topo I and topo II from the host cell.
2.1. Cells and viruses
* Corresponding author. Fax: + 1 (819) 5645392. 0167-4781/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 01 67-4781 ( 9 5 ) 0 0 0 5 0 - X
Baculovirus system: Sf9 cells, derived from an established cell line of Spodopterafrugiperda [13], were grown in suspension in Grave's medium supplemented with yeastolate and lactalbumin hydrolysate plus 10% fetal calf serum. Cells at a concentration of 2 . 1 0 6 per ml were infected with vEV51LT recombinant baculovirus [14] expressing Py LT. After 3 days of incubation at 27 ° C, the cells were lysed in buffer A, containing 20 mM Tris-HCl pH 8.0, 200 mM NaC1, 1 mM EDTA, 0.1% NP40, 1 mM DTI', 200 /~g/ml PMSF and 20% glycerol. The cell extract was centrifuged at 20 000 rpm for 30 min and the superuatant containing Py LT was stored at - 8 0 ° C until further use. Vaccinia virus system: HeLa cells were grown in suspension in DMEM added with 7% calf serum. The cells were infected (1 PFU/cell) with vaccinia virus recombinant VVpyLT ([15]; Transgene, 67082 Strasbourg C~dex) and incubated for 2 days at 37°C prior to being lysed as detailed above. 2.2. DNAs, enzymes, inhibitors and antibodies Purified pBR322 plasmid consisting mostly of form I DNA was purchased from Pharmacia. Calf thymus DNA
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A. Marton et al. / Biochimica et Biophysica Acta 1262 (1995) 59-63
topoisomerase I was obtained from Gibco/BRL. Camptothecin-lactone was provided by Nancita R. Lomax, National Cancer Institute (NIH, Bethesda). Monoclonal antibody LT-1 directed against Py LT [16] was generously provided by Beverly Griffin. Sera from scleroderma patients with high titers of autoantibodies against human topo I were a gift of Dr. Gilles Boire, Department of Rheumatology, University of Sherbrooke.
2.3. Purification of Py LT Py LT was purified from VVpyLT-infected HeLa cells or vEV51LT-infected Sf9 insect cells by immunopurification [3] followed by ion-exchange chromatography. The immunomatrix was prepared according to Schneider et al. [17] using LT-1, a rat monoclonal antibody, which was covalently attached to protein G-Sepharose (Pharmacia). After overnight incubation of the immunomatrix with the cell extract at 4° C, a packed column was made and washed sequentially with ten bed volumes of buffer B (50 mM Tris-HC1 pH 8.0, 500 mM NaCI, 1 mM EDTA, 1 mM DTT, 20% glycerol, 1% NP40), and then buffer C (same as buffer B, but adjusted to pH 9.0 and containing no NP40). The immunologically adsorbed LT was eluted with high pH buffer (same as buffer C but adjusted to pH 11) which, under our conditions, was found to be the most effective eluent. However, strong binding by the LT-1 antibody limited the recovery of LT to about 35 to 40%. After this first purification step, LT was about 80% pure, as judged from silver-staining after SDS-PAGE (Fig. 1, lane a). The fractions from the immunocolumn containing most of LT - as well as DNA relaxing activity - were
a
PyLT--
b
c
m
dialysed against buffer D (25 mM Tris-HC1 pH 8.0, 120 mM NaC1, 1 mM DTT, 1 mM EDTA, 20% glycerol) and then further purified on DEAE-Sephacel (Pharmacia) anion exchanger. Because of the sharp difference between the isoelectric points of Py LT and eukaryotic topo I (6.07 and 10.08, respectively), such ion-exchange chromatography was expected to provide further separation of LT from contaminating traces of the cellular enzyme. After extensive washing with buffer D, Py LT was eluted from the DEAE-Sephacel column with a 120-500 mM NaCI concentration gradient. At 200 mM salt concentration, LT started to come off from the column, allowing a 90% yield of homogeneous LT (Fig. 1, lane b, LT-vaccinia, lane c, LT-baculo). Finally, Py LT preparations were dialysed against 25 mM Tris-HCl pH 8.0, 1 mM DTT, 1 mM EDTA and 20% glycerol, before being stored at - 8 0 ° C.
2.4. DNA relaxation reactions 25 ng of negatively supercoiled pBR322 DNA were incubated for 30 min at 37°C in a total volume of 20 /zl with 50 ng of purified Py LT in buffer R. Buffer R contained 50 mM Tris-HC1 pH 8.0, 50 mM KC1, 1 mM DTT, 0.5 mM EDTA, 30 /xg/ml of nuclease-free BSA (Pharmacia) and 10 mM MgC12, unless indicated differently. Since the specific activity of LT-topo was rather low, comparisons with calf thymus topo I were made using similar activities rather than protein amounts. More specifically, we generally attempted to work with DNA/enzyme ratios allowing a 20 to 80% conversion of supercoiled DNA into relaxed DNA. Reactions were stopped by the addition of 0.5% SDS, followed by proteinase K treatment. The samples were then electrophoresed through a 1.2% agarose gel, blotted and hybridized with 32p-labelled pBR322 DNA prior to autoradiography.
3. Results
-94 kD -67 --43
Fig. 1. SDS-PAGE analysis of purified Py LT. Samples of LT were electrophoresed through a 10% SDS-polyacrylamide gel and then silverstained. (a) LT produced in the baculovirus virus system and immunopurifled; (b) the same LT after DEAE-Sephacel purification; (c) LT produced in the vaccinia virus system after immunopurification and DEAESephacel chromatography; m, molecular weight marker. In lane a, polypeptides migrating faster than Py LT are attributable to immunoglobulins released from the column.
3.1. Experimental design Whether isolated from insect or mammalian cells (Materials and methods), purified Py LT consisted almost exclusively of material migrating as a polypeptide of about 100 K during SDS-PAGE (Fig. 1, lanes b and c). All preparations displayed DNA-relaxing activity detectable either in the absence or in the presence of ATP (data not shown). In our previous work, we had shown that such LT-topo could neither be dissociated from SV40 LT nor generated by mixing SV40 LT with topo I [3]. Although such experiments apparently excluded that LT-topo could be due to a contaminating enzyme, this conclusion could not be definitive. Thus, in the case of Py LT, we decided to rely on experiments which would ask directly whether the cognate LT-topo was distinct from potential contaminant topoisomerases. All our experiments were carried out
A. Marton et al. / Biochimica et Biophysica Acta 1262 (1995) 59-63
C P T (mM) o lo 50 loo o lo 50 lOO
-II
-I a
b
c
d
I
e f I
cj
h
I
Topoi
I
LT-Topo
Fig. 2. inhibition of relaxation by camptothecin-lactone (CPT). Constant amounts of calf thymus topo I or LT were mixed with increasing amounts of CPT and the mixtures tested for their ability to relax pBR322 DNA. After stopping the reactions, the samples were loaded onto a 1% agarose gel before electrophoresis and DNA blotting. Electrophoresis was carried out here as described by Champoux and Aronoff [26] in order to separate nicked DNA (II) from relaxed covalently-closed DNA (R). I, negatively supercoiled DNA.
61
we investigated whether relaxation by LT-topo would be affected by camptothecin, which inhibits topo I but not topo II [22]. Such an experiment was expected to be doubly informative for Py LT produced in the vaccinia system, since the vaccinia genome encodes a topo I that is unique precisely for being resistant to camptothecin [23]. The effect of camptothecin-lactone on relaxation by LTtopo or calf thymus topo I was thus determined, both reactions being performed at low enzyme/DNA ratio. As expected [22], calf thymus topo I was inhibited by camptothecin-lactone, decreasing amounts of topoisomers being observed for increasing doses of the inhibitor (Fig. 2, lanes a-d). LT-topo was also inhibited by camptothecin-lactone, at least as effectively as topo I (Fig. 2, lanes e-h). Therefore, LT-topo responds to camptothecin like eukaryotic topo I does, but not like eukaryotic topo II or vaccinia virus topo I, two enzymes that might have co-purified with LT. 3.3. LT-topo is distinct from eukaryotic topo I
with Py LTs generated in the baculovirus and in the vaccinia virus system, with identical results. 3.2. LT-topo is distinct from both eukaryotic topo H and vaccinia topo I The activity of topo II is usually distinguished from that of topo I on the basis of two criteria: dependence on ATP and susceptibility to inhibitors [18,19]. That LT-topo would relax DNA in the absence of ATP (see above) already suggested that it was unrelated to topo II [20,21]. Hence,
A
B
S I
C
0
Sera from patients suffering from scleroderma (Materials and methods) and containing high titers of autoantibodies directed against human topo I were tested for their ability to interfere with DNA relaxation. As expected from topo I known conservation amongst eukaryotes and from previous data from other laboratories [24], such sera strongly inhibited relaxation by calf thymus topo I (Fig. 3A). However, the same sera had no effect on relaxation by LT-topo (Fig. 3B). This result (Fig. 3B), which was obtained with LT produced in the vaccinia system, argues
S
i
1
2
I
3
0
I
1
2
3
II+R
I
I
Topoi
I
I
LT-Topo (Vaccinia)
Fig. 3. Inhibition of DNA relaxation by serum from scleroderma patients. 2 /1.1of a l:1000 dilution of serum (S) from three different patients (Si, S 2, S 3) were mixed with topo I (A) or LT (B) and preincubated for 10 rain at 37 ° C before the reaction was started by adding pBR322 DNA to the mix. Relaxation was assessed as in Fig. 2 except that electrophoresis was run under standard conditions, which did not allow separation of nicked DNA (II) from relaxed covalently-closed DNA (R). Lanes labelled 0 were run with DNA treated with enzyme in the absence of antibodies, and lane C with DNA incubated without enzyme or antibody.
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A. Marton et al. / Biochimica et Biophysica Acta 1262 (1995) 59-63
A
B
EDTA(mM) MgCi2(rnM)
0 10
0 0
0 10 10 10
a
b
0 0
c
I
a I
0 10
10 10
b
c
I
Topo I
I
LT-Topo (Vaccinia)
Fig. 4. Mg 2+ dependence of DNA relaxation by LT-topo. pBR322 DNA was mixed with either calf thymus topo I (A) or Py LT (B) and incubated in a buffer containing neither EDTA nor MgCI 2 (lanes a), no EDTA and 10 mM MgC12 (lanes b), 10 mM EDTA and 10 mM MgC12 (lanes c). Relaxation was assessed as done in Fig. 3. The first lane in A was run with DNA incubated in the absence of either enzyme. Note that bands indicative of partially relaxed topoisomers are strongest in lane b for topo I (A), and absent from lanes a and c for LT-topo (B).
quite strongly against LT-topo being attributable to the topo I indigenous to the cells (HeLa) in which this vector had been expressed (see also Discussion).
3.4. Unlike eukaryotic topo L LT-topo requires Mg 2+ cations Relaxation by eukaryotic topo I is inherently Mg2÷-in dependent, even though the Mg 2÷ cation exerts on the
A
B No Mg 2+ I. . . . .
T0po
reaction a stimulatory effect that is discernible at low enzyme concentration [25]. In a first experiment, we investigated how amounts of calf thymus topo I and LT-topo allowing only partial DNA relaxation would be affected by a standard concentration of MgCI 2 (Fig. 4). Actually, topo I relaxed DNA under any condition, although it was more active in the presence of MgCI 2 (Fig. 4A, compare lane b with lanes a and c). As to LT-topo, DNA topoisomers were observed only when the reaction was carded out in
10mMMg I
f
No Mg 2+
2+ I
(U) o. ;. .~ . ~.. .o 2 ~~ ,o d , - :o
[
LT (ng)
10mMMg2÷ l
I
I
° 0 ~ ~ g °=,.- o,° ~ o© = ,.II+R
I
a bc
d e f Topoi
gh
i
a b c ci e LT-Topo
f g h
i
(Vaccinia)
Fig. 5. Mg 2+ dependence of DNA relaxation by LT-topo. pBR322 DNA was incubated with increasing amounts of topo I (A, units) or LT (B, nanograms) in either the absence or the presence of 10 mM MgCI2, and subsequently characterized as in Fig. 3. Note in B that the intensity of band I (or that of II + R) does not vary markedly when one moves from lanes a to e (no Mg 2÷ ), whereas band I progressively disappears between lanes f and i (10 mM Mg 2+ ), as expected if Mg 2÷ was absolutely required for enzyme activity.
A. Marton et al. / Biochimica et Biophysica Acta 1262 (1995) 59-63
the presence of MgC12 (Fig. 4B). In a second experiment, MgCI 2 concentration was kept constant at either 0 mM or 10 mM, and various concentrations of the enzymes were used. In the case of calf thymus topo I, relaxation again took place with or without MgCI 2 (Fig. 5A), being only stimulated by MgCI 2 at low enzyme concentration (Fig. 5A, compare lanes b and f). On the contrary, for LT-topo, no relaxation could be observed in the absence of MgC12 (Fig. 5B), even for enzyme concentrations causing total disappearance of form I DNA in the presence of Mg 2+ (Fig. 5B, compare lanes e and i). Thus, an absolute requirement for Mg 2+ ions characterizes DNA relaxation by Py LT-topo.
63
supposes a rather intimate and specific interaction between the two proteins.
Acknowledgements This work was supported by grants from the Medical Research Council (MRC) of Canada to P.B. and D. Bourgaux-Ramoisy, including a University-Industry grant funded jointly by MRC and Pharmacia Inc. Carol Prives was kind enough to provide us with vEV51LT, and M.P. Kieny (Transgene) with VVpyLT. Text editing by Andr6e Houle and Nicole Blais is gratefully acknowledged. We thank Raymund Wellinger for critical review of the manuscript.
4. Discussion References The data reported above indicate that purified Py LT obtained from two different sources displays a DNA relaxing activity exerting itself in the absence of a source of energy such as ATP. Whereas this activity resembles in some respects that of eukaryotic topo I, it does not appear to reflect the association of LT with the topo I originating from either the cells or the viral expression vector used to produce LT. This seems particularly clear for LT produced in the vaccinia virus system. As shown in Fig. 2, relaxation by such LT was inhibited by camptothecin-lactone, and thus could not be attributed to contaminating vaccinia virus topo I. Also, relaxation by the same LT preparation was not inhibited by human autoantibodies strongly inhibiting relaxation by calf thymus topo I (Fig. 3). Yet, we and others have found that sera from scleroderma patients neutralize topo I from animal cells ranging from D. melanogaster to H. sapiens ([3]; see Ref. [25] for a review). Thus, the relaxing activity of the LT produced in the vaccinia system could not be due to either of the topo I most likely to contaminate such preparations, HeLa topo I and vaccinia virus topo I. Given the known properties of LT, our results may indicate that LT either has intrinsic topoisomerase activity or associates with a yet undescribed, Mga+-dependent, cellular topoisomerase against which scleroderma patients do not produce antibodies. Both possibilities are of obvious interest, considering that it is the ability of papovavirus LT to associate with specific cellular proteins which led to the discovery of protein p53 [11,12]. In view of the ambivalence of our results, we should underline the Mg 2+ dependence of the LT-topo reaction. To our knowledge, this requirement is known to exist for prokaryotic but not for eukaryotic topo I [24]. It may thus indicate that LT-topo is intrinsic to Py LT, or that Py LT interacts with a yet undescribed cellular topoisomerase or lastly, that the binding of topo I to LT affects the reactivity and requirements of the former enzyme. Either of the two latter explanations
[1] DePamphilis, M.L. and Bradley, M.K. (1986) In The Papovaviridae, (Salzman, N.P., ed.), Vol. 1, pp. 99-246, Plenum Press, New York. [2] Mann, K. (1993) Nucleic Acids Res. 21, 1697-1704. [3] Marton, A., Jean, D., Delbecchi, L., Simmons, D.T. and Bourgaux, P. (1993) Nucleic Acids Res. 21, 1689-1695. [4] Liu, L.F. and Wang, J.C. (1987) Proc. Natl. Acad. Sci. USA 84, 7024-7027. [5] Nault, C., Veilleux, S., Delbecchi, L., Bourgaux-Ramoisy, D. and Bourgaux, P. (1994) Nucleic Acids Res. 22, 485-491. [6] Sadowski, P. (1986) J. Bacteriol. 165, 341-347. [7] Clayton, C.E., Murphy, D., Lovett, M. and Rigby, P.W.J. (1982) Nature 299, 59-61. [8] Tornow, J. and Cole, C.N. (1983) Proc. Natl. Acad. Sci. USA 80, 6312-6316. [9] Cole, C.N., Crawford, L.V. and Berg, P. (1979) J. Virol. 30, 683 -691. [10] Tooze, J. (1980) Molecular Biology of Tumor Viruses, Vol. 2, Cold Spring Harbor Laboratory Press, Cold Spring Harbor. [1 l] Lane, D.P. and Crawford, L.V. (1979) Nature 278, 261-263. [12] Linzer, D.I.H. and Levine, A.J. (1979) Cell 17, 43-52. [13] Vaughn, J.L., Goodwin, R.H., Thompkins, G.L. and McCawley, P. (1977) In Vitro 13, 213-217. [14] Rice, W.C., Lorimer, H.E., Prives, C. and Miller, L.K. (1987) J. Virol. 61, 1712-1716. [15] Lathe, R., Kieny, M.P., Gerlinger, P., Clertant, P., Guizani, I., Cuzin, F. and Chambon, P. (1987) Nature 326, 878-880. [16] Dilworth, S.T. and Griffin, B. (1982) Proc. Natl. Acad. Sci. USA 79, 1059-1063. [17] Schneider, C., Newman, R.A., Sutherland, D.R., Asser, U. and Greaves, M.F. (1982) J. Biol. Chem. 257, 10766-10769. [18] Drlica, K. and Franco, R.J. (1988) Biochemistry 27, 2253-2259. [19] Liu, L.F. (1989) Annu. Rev. Biochem. 58, 351-375. [20] Goto, T. and Wang, J.C. (1982) J. Biol. Chem. 257, 5866-5872. [21] Miller, K.G., Liu, L.F. and Englund, P.T. (1981) J. Biol. Chem. 256, 9334-9339. [22] Hsiang, Y.-H., Hertzberg, R., Hecht, S. and Liu, L.F. (1985) J. Biol. Chem. 260, 14873-14878. [23] Shuman, S., Golder, M. and Moss, B. (1988) J. Biol. Chem. 263, 16401 - 16407. [24] Piccinini, G., Cardellini, E., Reimer, G., Aruett, F.C. and Durban, E. (1991) Mol. Immunol. 28, 333-339. [25] Wang, J.C. (1985) Annu. Rev. Biochem. 54, 665-697. [26] Champoux, J.J. and Aronoff, R. (1989) J. Biol. Chem. 264, 10101015.
B iochimica et Biophysica Acta 1262 (1995) 59-63
Biochim~ic~a et BiophysicaA~ta
Topoisomerase activity associated with polyoma virus large tumor antigen Attila Marton, Bernadette Marko, Louis Delbecchi, Pierre Bourgaux
*
Department of Microbiology, Faculty of Medicine, Universit~ de Sherbrooke, 3001, 12e Avenue Nord, Sherbrooke, Quebec, J1H 5N4, Canada Received 24 November 1994; accepted 22 February 1995
Abstract
Polyomavirus (Py) large tumor antigen (LT) was produced in mammalian or insect cells infected with a suitable viral expression vector, and purified by a procedure combining immunoprecipitation with ion-exchange chromatography. Fractions containing the bulk of LT displayed a DNA-relaxing activity (LT-topo) which could be attributed neither to topoisomerase II (topo II) nor to topoisomerase I (topo I) encoded by the cell or the viral vector. On the one hand, LT-topo relaxed pBR322 DNA in a reaction which, unlike that characteristic of topo II, was ATP-independent and inhibited by camptothecin. On the other hand, serum from scleroderma patients which strongly inhibited calf thymus topo I had no effect on LT-topo, which absolutely required Mg 2+ ions to relax DNA. Thus, LT-topo is either inherent to LT or belongs to a LT-bound enzyme similar to, but distinct from, topo I. Keywords: Polyoma large T antigen; DNA relaxation; Camptothecin; Scleroderma; Magnesium ion-dependent topoisomerase
1. Introduction
2. Materials and methods
The large tumor antigens (LTs) of SV40 and polyoma virus (Py) are multifunctional proteins known to interact specifically with DNA as well as with cellular proteins [1]. Recently, we and others [2,3] have shown that purified preparations of SV40 LT have topoisomerase activity, a finding of potential significance with respect to the role of LT in replication [1], transcription [4] and recombination [5,6]. Available data do not allow us to decide whether this activity was inherent to or associated with SV40 LT, even though trivial contamination could be clearly excluded [2,3]. Py LT is structurally and functionally similar but not identical to SV40 LT. For example, SV40 LT carries a transforming function [7,8] and a function allowing the complementation of adenovirus [9], both of which are absent from Py LT [10]. Also, SV40 LT, but not Py LT, interacts with the host protein p53 [11,12]. It was thus conceivable that purified Py LT would lack the DNA-relaxing activity associated with SV40 LT. We show here that preparations of Py LT display a DNA-relaxing activity (LT-topo) similar to that of SV40 LT [2,3], but clearly distinct from those of potential contaminants such as topo I and topo II from the host cell.
2.1. Cells and viruses
* Corresponding author. Fax: + 1 (819) 5645392. 0167-4781/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 01 67-4781 ( 9 5 ) 0 0 0 5 0 - X
Baculovirus system: Sf9 cells, derived from an established cell line of Spodopterafrugiperda [13], were grown in suspension in Grave's medium supplemented with yeastolate and lactalbumin hydrolysate plus 10% fetal calf serum. Cells at a concentration of 2 . 1 0 6 per ml were infected with vEV51LT recombinant baculovirus [14] expressing Py LT. After 3 days of incubation at 27 ° C, the cells were lysed in buffer A, containing 20 mM Tris-HCl pH 8.0, 200 mM NaC1, 1 mM EDTA, 0.1% NP40, 1 mM DTI', 200 /~g/ml PMSF and 20% glycerol. The cell extract was centrifuged at 20 000 rpm for 30 min and the superuatant containing Py LT was stored at - 8 0 ° C until further use. Vaccinia virus system: HeLa cells were grown in suspension in DMEM added with 7% calf serum. The cells were infected (1 PFU/cell) with vaccinia virus recombinant VVpyLT ([15]; Transgene, 67082 Strasbourg C~dex) and incubated for 2 days at 37°C prior to being lysed as detailed above. 2.2. DNAs, enzymes, inhibitors and antibodies Purified pBR322 plasmid consisting mostly of form I DNA was purchased from Pharmacia. Calf thymus DNA
60
A. Marton et al. / Biochimica et Biophysica Acta 1262 (1995) 59-63
topoisomerase I was obtained from Gibco/BRL. Camptothecin-lactone was provided by Nancita R. Lomax, National Cancer Institute (NIH, Bethesda). Monoclonal antibody LT-1 directed against Py LT [16] was generously provided by Beverly Griffin. Sera from scleroderma patients with high titers of autoantibodies against human topo I were a gift of Dr. Gilles Boire, Department of Rheumatology, University of Sherbrooke.
2.3. Purification of Py LT Py LT was purified from VVpyLT-infected HeLa cells or vEV51LT-infected Sf9 insect cells by immunopurification [3] followed by ion-exchange chromatography. The immunomatrix was prepared according to Schneider et al. [17] using LT-1, a rat monoclonal antibody, which was covalently attached to protein G-Sepharose (Pharmacia). After overnight incubation of the immunomatrix with the cell extract at 4° C, a packed column was made and washed sequentially with ten bed volumes of buffer B (50 mM Tris-HC1 pH 8.0, 500 mM NaCI, 1 mM EDTA, 1 mM DTT, 20% glycerol, 1% NP40), and then buffer C (same as buffer B, but adjusted to pH 9.0 and containing no NP40). The immunologically adsorbed LT was eluted with high pH buffer (same as buffer C but adjusted to pH 11) which, under our conditions, was found to be the most effective eluent. However, strong binding by the LT-1 antibody limited the recovery of LT to about 35 to 40%. After this first purification step, LT was about 80% pure, as judged from silver-staining after SDS-PAGE (Fig. 1, lane a). The fractions from the immunocolumn containing most of LT - as well as DNA relaxing activity - were
a
PyLT--
b
c
m
dialysed against buffer D (25 mM Tris-HC1 pH 8.0, 120 mM NaC1, 1 mM DTT, 1 mM EDTA, 20% glycerol) and then further purified on DEAE-Sephacel (Pharmacia) anion exchanger. Because of the sharp difference between the isoelectric points of Py LT and eukaryotic topo I (6.07 and 10.08, respectively), such ion-exchange chromatography was expected to provide further separation of LT from contaminating traces of the cellular enzyme. After extensive washing with buffer D, Py LT was eluted from the DEAE-Sephacel column with a 120-500 mM NaCI concentration gradient. At 200 mM salt concentration, LT started to come off from the column, allowing a 90% yield of homogeneous LT (Fig. 1, lane b, LT-vaccinia, lane c, LT-baculo). Finally, Py LT preparations were dialysed against 25 mM Tris-HCl pH 8.0, 1 mM DTT, 1 mM EDTA and 20% glycerol, before being stored at - 8 0 ° C.
2.4. DNA relaxation reactions 25 ng of negatively supercoiled pBR322 DNA were incubated for 30 min at 37°C in a total volume of 20 /zl with 50 ng of purified Py LT in buffer R. Buffer R contained 50 mM Tris-HC1 pH 8.0, 50 mM KC1, 1 mM DTT, 0.5 mM EDTA, 30 /xg/ml of nuclease-free BSA (Pharmacia) and 10 mM MgC12, unless indicated differently. Since the specific activity of LT-topo was rather low, comparisons with calf thymus topo I were made using similar activities rather than protein amounts. More specifically, we generally attempted to work with DNA/enzyme ratios allowing a 20 to 80% conversion of supercoiled DNA into relaxed DNA. Reactions were stopped by the addition of 0.5% SDS, followed by proteinase K treatment. The samples were then electrophoresed through a 1.2% agarose gel, blotted and hybridized with 32p-labelled pBR322 DNA prior to autoradiography.
3. Results
-94 kD -67 --43
Fig. 1. SDS-PAGE analysis of purified Py LT. Samples of LT were electrophoresed through a 10% SDS-polyacrylamide gel and then silverstained. (a) LT produced in the baculovirus virus system and immunopurifled; (b) the same LT after DEAE-Sephacel purification; (c) LT produced in the vaccinia virus system after immunopurification and DEAESephacel chromatography; m, molecular weight marker. In lane a, polypeptides migrating faster than Py LT are attributable to immunoglobulins released from the column.
3.1. Experimental design Whether isolated from insect or mammalian cells (Materials and methods), purified Py LT consisted almost exclusively of material migrating as a polypeptide of about 100 K during SDS-PAGE (Fig. 1, lanes b and c). All preparations displayed DNA-relaxing activity detectable either in the absence or in the presence of ATP (data not shown). In our previous work, we had shown that such LT-topo could neither be dissociated from SV40 LT nor generated by mixing SV40 LT with topo I [3]. Although such experiments apparently excluded that LT-topo could be due to a contaminating enzyme, this conclusion could not be definitive. Thus, in the case of Py LT, we decided to rely on experiments which would ask directly whether the cognate LT-topo was distinct from potential contaminant topoisomerases. All our experiments were carried out
A. Marton et al. / Biochimica et Biophysica Acta 1262 (1995) 59-63
C P T (mM) o lo 50 loo o lo 50 lOO
-II
-I a
b
c
d
I
e f I
cj
h
I
Topoi
I
LT-Topo
Fig. 2. inhibition of relaxation by camptothecin-lactone (CPT). Constant amounts of calf thymus topo I or LT were mixed with increasing amounts of CPT and the mixtures tested for their ability to relax pBR322 DNA. After stopping the reactions, the samples were loaded onto a 1% agarose gel before electrophoresis and DNA blotting. Electrophoresis was carried out here as described by Champoux and Aronoff [26] in order to separate nicked DNA (II) from relaxed covalently-closed DNA (R). I, negatively supercoiled DNA.
61
we investigated whether relaxation by LT-topo would be affected by camptothecin, which inhibits topo I but not topo II [22]. Such an experiment was expected to be doubly informative for Py LT produced in the vaccinia system, since the vaccinia genome encodes a topo I that is unique precisely for being resistant to camptothecin [23]. The effect of camptothecin-lactone on relaxation by LTtopo or calf thymus topo I was thus determined, both reactions being performed at low enzyme/DNA ratio. As expected [22], calf thymus topo I was inhibited by camptothecin-lactone, decreasing amounts of topoisomers being observed for increasing doses of the inhibitor (Fig. 2, lanes a-d). LT-topo was also inhibited by camptothecin-lactone, at least as effectively as topo I (Fig. 2, lanes e-h). Therefore, LT-topo responds to camptothecin like eukaryotic topo I does, but not like eukaryotic topo II or vaccinia virus topo I, two enzymes that might have co-purified with LT. 3.3. LT-topo is distinct from eukaryotic topo I
with Py LTs generated in the baculovirus and in the vaccinia virus system, with identical results. 3.2. LT-topo is distinct from both eukaryotic topo H and vaccinia topo I The activity of topo II is usually distinguished from that of topo I on the basis of two criteria: dependence on ATP and susceptibility to inhibitors [18,19]. That LT-topo would relax DNA in the absence of ATP (see above) already suggested that it was unrelated to topo II [20,21]. Hence,
A
B
S I
C
0
Sera from patients suffering from scleroderma (Materials and methods) and containing high titers of autoantibodies directed against human topo I were tested for their ability to interfere with DNA relaxation. As expected from topo I known conservation amongst eukaryotes and from previous data from other laboratories [24], such sera strongly inhibited relaxation by calf thymus topo I (Fig. 3A). However, the same sera had no effect on relaxation by LT-topo (Fig. 3B). This result (Fig. 3B), which was obtained with LT produced in the vaccinia system, argues
S
i
1
2
I
3
0
I
1
2
3
II+R
I
I
Topoi
I
I
LT-Topo (Vaccinia)
Fig. 3. Inhibition of DNA relaxation by serum from scleroderma patients. 2 /1.1of a l:1000 dilution of serum (S) from three different patients (Si, S 2, S 3) were mixed with topo I (A) or LT (B) and preincubated for 10 rain at 37 ° C before the reaction was started by adding pBR322 DNA to the mix. Relaxation was assessed as in Fig. 2 except that electrophoresis was run under standard conditions, which did not allow separation of nicked DNA (II) from relaxed covalently-closed DNA (R). Lanes labelled 0 were run with DNA treated with enzyme in the absence of antibodies, and lane C with DNA incubated without enzyme or antibody.
62
A. Marton et al. / Biochimica et Biophysica Acta 1262 (1995) 59-63
A
B
EDTA(mM) MgCi2(rnM)
0 10
0 0
0 10 10 10
a
b
0 0
c
I
a I
0 10
10 10
b
c
I
Topo I
I
LT-Topo (Vaccinia)
Fig. 4. Mg 2+ dependence of DNA relaxation by LT-topo. pBR322 DNA was mixed with either calf thymus topo I (A) or Py LT (B) and incubated in a buffer containing neither EDTA nor MgCI 2 (lanes a), no EDTA and 10 mM MgC12 (lanes b), 10 mM EDTA and 10 mM MgC12 (lanes c). Relaxation was assessed as done in Fig. 3. The first lane in A was run with DNA incubated in the absence of either enzyme. Note that bands indicative of partially relaxed topoisomers are strongest in lane b for topo I (A), and absent from lanes a and c for LT-topo (B).
quite strongly against LT-topo being attributable to the topo I indigenous to the cells (HeLa) in which this vector had been expressed (see also Discussion).
3.4. Unlike eukaryotic topo L LT-topo requires Mg 2+ cations Relaxation by eukaryotic topo I is inherently Mg2÷-in dependent, even though the Mg 2÷ cation exerts on the
A
B No Mg 2+ I. . . . .
T0po
reaction a stimulatory effect that is discernible at low enzyme concentration [25]. In a first experiment, we investigated how amounts of calf thymus topo I and LT-topo allowing only partial DNA relaxation would be affected by a standard concentration of MgCI 2 (Fig. 4). Actually, topo I relaxed DNA under any condition, although it was more active in the presence of MgCI 2 (Fig. 4A, compare lane b with lanes a and c). As to LT-topo, DNA topoisomers were observed only when the reaction was carded out in
10mMMg I
f
No Mg 2+
2+ I
(U) o. ;. .~ . ~.. .o 2 ~~ ,o d , - :o
[
LT (ng)
10mMMg2÷ l
I
I
° 0 ~ ~ g °=,.- o,° ~ o© = ,.II+R
I
a bc
d e f Topoi
gh
i
a b c ci e LT-Topo
f g h
i
(Vaccinia)
Fig. 5. Mg 2+ dependence of DNA relaxation by LT-topo. pBR322 DNA was incubated with increasing amounts of topo I (A, units) or LT (B, nanograms) in either the absence or the presence of 10 mM MgCI2, and subsequently characterized as in Fig. 3. Note in B that the intensity of band I (or that of II + R) does not vary markedly when one moves from lanes a to e (no Mg 2÷ ), whereas band I progressively disappears between lanes f and i (10 mM Mg 2+ ), as expected if Mg 2÷ was absolutely required for enzyme activity.
A. Marton et al. / Biochimica et Biophysica Acta 1262 (1995) 59-63
the presence of MgC12 (Fig. 4B). In a second experiment, MgCI 2 concentration was kept constant at either 0 mM or 10 mM, and various concentrations of the enzymes were used. In the case of calf thymus topo I, relaxation again took place with or without MgCI 2 (Fig. 5A), being only stimulated by MgCI 2 at low enzyme concentration (Fig. 5A, compare lanes b and f). On the contrary, for LT-topo, no relaxation could be observed in the absence of MgC12 (Fig. 5B), even for enzyme concentrations causing total disappearance of form I DNA in the presence of Mg 2+ (Fig. 5B, compare lanes e and i). Thus, an absolute requirement for Mg 2+ ions characterizes DNA relaxation by Py LT-topo.
63
supposes a rather intimate and specific interaction between the two proteins.
Acknowledgements This work was supported by grants from the Medical Research Council (MRC) of Canada to P.B. and D. Bourgaux-Ramoisy, including a University-Industry grant funded jointly by MRC and Pharmacia Inc. Carol Prives was kind enough to provide us with vEV51LT, and M.P. Kieny (Transgene) with VVpyLT. Text editing by Andr6e Houle and Nicole Blais is gratefully acknowledged. We thank Raymund Wellinger for critical review of the manuscript.
4. Discussion References The data reported above indicate that purified Py LT obtained from two different sources displays a DNA relaxing activity exerting itself in the absence of a source of energy such as ATP. Whereas this activity resembles in some respects that of eukaryotic topo I, it does not appear to reflect the association of LT with the topo I originating from either the cells or the viral expression vector used to produce LT. This seems particularly clear for LT produced in the vaccinia virus system. As shown in Fig. 2, relaxation by such LT was inhibited by camptothecin-lactone, and thus could not be attributed to contaminating vaccinia virus topo I. Also, relaxation by the same LT preparation was not inhibited by human autoantibodies strongly inhibiting relaxation by calf thymus topo I (Fig. 3). Yet, we and others have found that sera from scleroderma patients neutralize topo I from animal cells ranging from D. melanogaster to H. sapiens ([3]; see Ref. [25] for a review). Thus, the relaxing activity of the LT produced in the vaccinia system could not be due to either of the topo I most likely to contaminate such preparations, HeLa topo I and vaccinia virus topo I. Given the known properties of LT, our results may indicate that LT either has intrinsic topoisomerase activity or associates with a yet undescribed, Mga+-dependent, cellular topoisomerase against which scleroderma patients do not produce antibodies. Both possibilities are of obvious interest, considering that it is the ability of papovavirus LT to associate with specific cellular proteins which led to the discovery of protein p53 [11,12]. In view of the ambivalence of our results, we should underline the Mg 2+ dependence of the LT-topo reaction. To our knowledge, this requirement is known to exist for prokaryotic but not for eukaryotic topo I [24]. It may thus indicate that LT-topo is intrinsic to Py LT, or that Py LT interacts with a yet undescribed cellular topoisomerase or lastly, that the binding of topo I to LT affects the reactivity and requirements of the former enzyme. Either of the two latter explanations
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