Eur. J. Biochem. 210,359-364 (1992) 0FEBS 1992

Comparison of biochemical properties of DNA-topoisomerase I from normal and regenerating liver Marie-FranCoise TOURNIER

Joelle SOBCZAK', Beatrice de NECHAUD and Michel DUGUET '

Laboratoire d'Enzymologie des acides nucleiques, Universite Pierre et Marie Curie, Unite associee au Centre National de la Recherche Scientifique n c 554, Paris, France Inserm U75, Paris, France Laboratoire de Biochimie cellulaire, College de France, Paris, France (Received May 20/August 11, 1992) - EJB 92 0702

Biochemical properties of topoisomerase I from normal and regenerating rat liver were analysed using crude or fractionated nuclear extracts. We could not detect significative change in topoisomerase I content or activity (magnesium stimulation and inhibition by ATP) during the course of liver regeneration. Topoisomerase I can be resolved into two species of 97 kDa and 100 kDa, with the same p l o f 8.2 - 8.6 as shown by two dimensional gel electrophoresis. The two polypeptides contained a non-phosphorylated precursor and others forms with variable degrees of phosphorylation. In-vitro dephosphorylation with alkaline phosphatase leads to the disappearance of the phosphorylated forms and inactivation of the enzyme. The affinity of topoisomerase I for chromatin (measured by salt elution) differs markedly between normal and regenerating liver: nearly 50% of topoisomerase I remained bound to the chromatin from normal liver at 250 mM NaCl whereas it was completely eluted from 24-h-regenerating-liver nuclei. The biological significance of these results is discussed.

DNA topoisomerases are enzymes that can modify the topological state of DNA. Several data show that they are involved in DNA unwinding during transcription and replication (reviewed by Wang, 1985,1987). In vitro, topoisomerase I relaxes closed-circular double-stranded DNA by introducing a transient single-strand break and passing the intact strand through this break. This activity is abolished in vitro by dephosphorylation with calf-intestine alkaline phosphatase and can be subsequently reactivated by phosphorylation with either casein-kinase 11 or protein-kinase C (Durban et al., 1983, 1985; Kaiserman et al., 1988; Samuels et al., 1989; Pommier et al., 1990). In contrast, in-vitro phosphorylation of topoisomerase I by tyrosine kinases TPK 75 or pp60"'" leads to its inactivation (Tse-Dinh et al., 1984). These different data suggest that topoisomerase I activity could be regulated in vivo by phosphorylation/dephosphorylation. In addition, topoisomerase I was shown to be inactivated by ADPribosylation (Ferro and Olivera, 1984) and stimulated by interaction with histone H I or high-mobility-group proteins (Javaherian and Liu, 1983). Finally, ATP appears to act as an inhibitor of topoisomerase-I activity (Low and Holden, 1985; Chen and Castora, 1988) although this point is disputed (Goto et al., 1984; Coderoni et al., 1990). Regenerating liver is a refined model for studying in vivo the specific activation of genes and enzymes involved in the cell cycle. Hepatocytes enter the cell cycle synchronously, with a peak of DNA synthesis at 24 h post-hepatectomy and a peak Correspondence to M.-F. Tournier, Institut Jacques Monod, Genttique du developpement et evolution, Tour 42-32, 5e titage, 2 place Jussieu, F-75251 Paris Cedex 05, France Abbreviations. NEPHGE, non-equilibrium pH gradient electrophoresis.

of mitosis at 30 h post-hepatectomy (Michalopoulos,l990; Mohn et al., 1990). Eukaryotic type-I topoisomerase was first purified from rat liver (Champoux and Dulbecco, 1972). This liver enzyme exhibited a molecular mass of 64-68 kDa (Champoux and McConaughy, 1976; Been and Champoux, 1981) but a larger form was later reported (McConaughy et al., 1981). N o change in topoisomerase-I activity was detected during the regeneration process (Duguet et al., 1983) although topoisomerase-I-encoding mRNA was increased (Sobczak et al., 1989). The recent availability of antibodies specific for human topoisomerase I prompted us to reinvestigate the status of this protein during liver regeneration. In this study, we show the presence, in normal and regenerating liver, of two phosphorylated topoisomerase-I variants of molecular mass 97 - 100 kDa. The amount and enzymic properties of these variants is unchanged during the cell cycle in regenerating liver. Moreover, we show that, in normal liver, a fraction of topoisomerase I remains attached to the nuclear matrix while the enzyme can be completely eluted from regeneratingliver nuclei.

EXPERIMENTAL PROCEDURES Animals and labeling of DNA Male wistar rats (two-months old, 200 - 300 g) were anaesthetized under ether and subjected to partial hepatectomy (Higgins & Anderson,l931). Animals were fasted for 12 h before sacrifice and killed at various times after hepatectomy between 8 a.m. and 11 a.m., in order to avoid nycthemeral fluctuations. Liver was perfused with NaCl/Cit. (150 mM NaC1, 15 mM Sodium citrate, pH 7) before its isolation. 5-7 animals were used for each time point. DNA

360 synthesis was assessed by incorporation of [3H]thymidine (560 GBqjmmol), injected intraperitoneally (185 kBq/g animal) 2 h before the sacrifice. The percentage of cells in S phase was also determined by autoradiography of liver sections. Preparation of nuclear and cytoplasmic extracts Nuclear and cytoplasmic extracts were prepared at 4 "C by the method described by McConaughy et al. (1981) modified as follows. Minced livers were homogenized in a Potter with 10 vol. buffer A (10 mM Tris/HCl, pH 7.5, 0.25 M sucrose, 10 mM KC1, 5 mM MgCI2, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM dithiotreitol, 1 pg/ml pepstatin, 1 pg/ml leupeptin, 1 pg/ml aprotinin). The homogenate was centrifuged at 3000 g for 10 min. Triton XI00 (0.5% final), 0.1 vol. 200mM EGTA, 0.1 vol. 50mM EDTA and poly(ethy1ene glycol) 6000 (6% final) were successively added to the supernatant. The mixture was stirred for 45 rnin and then centrifuged at 12000 g for 60 min. Aliquots of the supernatant were frozen in liquid nitrogen and stored at - 80 "C for further use. These fractions were referred to as cytoplasmic. The nuclear pellet was resuspended in approximately 1 vol. 2.1 M sucrose with 10 mM Tris/HCl, pH 7.5,10 mM KCl and 5 mM MgCl,. After centrifugation at 48000 g for 30 min, the nuclear pellet was washed once with buffer A and once with the same buffer plus Triton XI00 (0.5%) and was then processed as indicated. Solubilization of proteins from the nuclear matrices Nuclear matrices were extracted in increasing salt concentrations essentially as described by Hancock (1974). The nuclear pellet was resuspended in buffer B (50 mM Tris/HCl, pH 7.5, 0.25 M sucrose, 10 mM KCI, 5 mM MgC12, 0.4 mM phenylmethylsulfonyl fluoride, 1 mM dithiotreitol and 1 pg/ ml pepstatin, 1 pg/ml leupeptin and 1 pg/ml aprotinin) at a concentration of 2 x lo8 nuclei/ml. 1 vol. elution buffer (10500 mM NaC1, 50 mM Tris/HCl, pH 7.5, 20% glycerol) was added. The suspension was rotated for 15 min and the nuclei were pelleted. The supernatant was referred to as soluble fraction. The pellet was resuspended and sonicated in Laemmli buffer and referred to as matrix fraction. The total nuclear extract was obtained by lysing the nuclei with elution buffer containing 2 M NaC1,20 mM EGTA and 5 mM EDTA. Nucleic acids were precipitated with 6% poly(ethy1ene glycol) 6000 and pelleted at 18000 g for 20 min. Aliquots of these different fractions were frozen in liquid nitrogen and stored at - 80 "C. Quantitative determination of proteins was performed according to Schaffner and Weissmann (1973) using bovine serum albumin as standard. Immunoblot analysis of topoisomerase I Proteins were separated on 0.1 Yo SDS/7.50/, polyacrylamide gels according to the procedure of Laemmli (1970). Prestained proteins (BRL) were used as molecular-mass markers. Transfer to nitrocellulose filters was performed in Tris/glycine/SDS buffer with 20% methanol (Towbin et a1.J 979). The filters were saturated with 3% fatty-acid-free milk powder and 1% bovine serum albumin and incubated with polyclonal rabbit (Oddou et al., 1988) or human scleroderma (Verheijen et al., 1990) anti-(topoisomerase I) sera at dilutions of 1: 50 and 1 :500, respectively during 1 h at room temperature. The rabbit polyclonal antiserum was raised against recombinant human topoisomerase I expressed in Escherichia coli from the pEV-vrf vector (Oddou et al., 1988).

The secondary antibody was either an alkaline phosphataseconjugated goat anti-(rabbit IgG) or anti-(human IgG) (1 : 5000; Calbiochem) and the color development was obtained with 5-bromo-4-chloro-3-indolyl phosphate (Sigma) and Nitro Blue Tetrazolium (Sigma) in alkaline phosphatase buffer. DNA-topoisomerase-I assay DNA-relaxation activity was assayed by agarose(Indubiose A37 NA, 1BF)-gel electrophoresis to monitor the relaxation of supercoiled (form I) pUC 9 plasmid DNA according to Duguet et a]., 1983. The assay mixture (15 pl) contained 15 mM Tris/HCl, pH 7.5, 0.2 mM EDTA, 0.5 mM dithiotreitol, 1YOglycerol, nuclease-free bovine serum albumin (10 pg/ml), pUC 9 DNA (250 ng) and proteins extracted from 100- 1500 nuclei. KCl or MgC12 were added at the concentration indicated in the legends to the figures. Each experiment, performed with four enzyme dilutions, was repeated twice. Reactions were incubated for 30 rnin at 37"C, stopped by the addition of l j 5 vol. EDTA 10 mM, SDS 0.5%, Bromophenol Blue 0.005°/~and glycerol 10%. The topoisomers were separated by electrophoresis through a 1.2% agarose gel at 9.6 V/cm for 2.2 h in buffer C (36 mM Tris, 1 mM EDTA, 30 mM NaH2P04, pH 7.8) with recirculation of the buffer. The photographic negative of each gel was acquired with a CCD camera driven by Azur software (ORKIS, France). Excel software (Microsoft) was used for further analysis. The activity was quantified as the disappearance of supercoiled DNA (expressed as the percentage of relaxed DNA in each sample). The proportion of form I1 (circular nicked form) was substracted from each group of assays. 1 U topoisomerase I was defined as the amount of enzyme necessary to relax 50% of supercoiled DNA in 30 min at 37 "C. Dephosphorylation of liver nuclear extract Insoluble calf-intestine alkaline phosphatase attached to beaded agarose (Sigma) was washed with water and resuspended (0.05 Ujpl) in phosphatase buffer (Pommier et al., 1990) with 1 mM dithiotreitol and protease inhibitors. Proteins from 5 x lo6- lo7 nuclei were incubated at 37°C for 30 min with 20 U alkaline phosphatase.

Two dimensional gel electrophoresis Separation of topoisomerase-I variants by two-dimensional gel electrophoresis was performed as described by O'Farrell et al. (1977). Proteins were first separated by the non-equilibrium pH gradient electrophoresis (NEPHGE) in the pH range 4-9.5, in a 12-cm cylindrical gel of 2.5 mm diameter. Electrophoresis in the first dimension was achieved for 5 h at 500 V at room temperature. The pH gradient obtained by this procedure was determined by slicing two reference gels run in parallel. 5-mm-long slices were soaked overnight in 500 pl H,O; the pH of the supernatant was measured at room temperature. The second dimension was performed in a 0.1% SDS/7.5% polyacrylamide gel of 21-cm length run at 60 V overnight followed by 5 h at 200 V. Transfer procedure on nitrocellulose was as described above.

RESULTS Enzyme activity of topoisomerase I is unchanged during the regenerating process We have prepared crude nuclear extracts from normal and regenerating liver at different times post-hepatectomy ; 16 h,

361 Table 1. Levels of DNA-topoisomerase-I activity in normal and regenerating liver under different ionic conditions. The enzyme activity was calculated from three serial dilutions of each extract. Values in parentheses represent the number of nuclei counted on liver sections. Time after partial Hepatectomy

Total nuclear proteins

Cells in S phase

h

pg/106 nuclei

%

lo3 cpm/mg DNA

u/105 nuclei

u/pg protein

u/105 nuclei

u/pg protein

0, Normal liver 16 24 30

9.3 11.1 6.8 9.3

0.9(4310) 0.2(4800) 35.1 ( 1 900) 17.0(904)

25.5 14.5 262.2 38.3

311 347 354 319

334 312 514 344

500 679 549 512

537 612 807 548

[3H]Thymidine incorporation in nuclei

Topoisomerase-I activity in 250 mM KCI

200 mM KC1/5 mM MgCI2

1

100

2

3

4

195,

80 Q

z

n

60

n al X

-2 s

40

105C

20

71,

0

I

0

1

2

3

4

5

6

7

8

9

10

.,

ATP (mM)

Fig. 1. Effects of ATP on the relaxation activity of normal-liver nuclear extract. Assays were performed with 875 pg total nuclear proteins 250 mM KCl; 0,200 mM KCl/ in various ionic conditions. 5 mM MgC12; A , 50 mM KClj20 mM MgC12.

representing the end of the G1 phase; 24 h, representing the S phase (Table 1); 30 h, representing mitosis at the end of the hepatocytes first cell cycle. The results of the enzymic assays in 250 mM KCl indicate that topoisomerase-I activity remains unchanged during the regeneration process (Table 1). Moreover, stimulation of topoisomerase-I activity with Mg2 (5 mM) in high concentration of KC1 (200 mM) was found identical in each extract (Table 1). The effect of ATP was also investigated. Fig. 1 shows that topoisomerase I from normal liver is inhibited by ATP. In the presence of high (250 mM) K C concentration, about 90% inhibition of DNA relaxation is observed with 10 mM ATP. Significant inhibition is also detected with lower concentrations of ATP, down to 2mM. The same effect was seen in high (200 mM) K f concentration in the presence of Mg2+ (5 mM) although higher concentrations of ATP (> 6 mM) were required to observe a significant inhibition. In low (50 mM) K f concentration, in the presence of Mg2+ (20mM), only a slight inhibitory effect was observed with increasing amounts of ATP. The same results were obtained with the various regenerating liver extracts (data not shown). +

Existence of two phosphorylated variants of topoisomerase I in normal and regenerating rat liver Analysis by western blot Total nuclear extracts from normal-liver and regeneratingliver nuclei were compared on the same blot. Two polypeptides

Fig. 2. Comparison of topoisomerase-I patterns in quiescent and proliferative liver cells by Western-blot analysis, using a scleroderma anti(topoisomerase I) serum. Some additional minor bands are visible; these bands are presumably aspecific since they are not found with the other antiserum. Proteins from 5 x lo6 nuclei were loaded in each lane. Lane 1, normal liver; lane 2,16 h post-hepatectomy; lane 3,24 h post-hepatectomy ;lane 4, 30 h post-hepatectomy. Molecular masses, in kDa, are indicated on the left.

reacted with the human scleroderma anti-(topoisomerase I) serum (Fig. 2) as well as with the antiserum directed against a cloned fragment of human topoisomerase I (see also Fig. 4). The apparent molecular mass of the two topoisomerase-I variants detected in this experiment is respectively 97 kDa and 100 kDa. These two molecular-mass variants are present in almost equimolar amount. Their total amount in each nucleus did not increase significantly during the regenerating process (only 1.3-1.5-fold over the normal liver value). The previously described 67-kDa form was barely detectable, even after the alkaline phosphatase reaction was completed (see also Figs 4 and 5). The two topoisomerase-I variants are phosphorylated To further characterize the two topoisomerase-I variants, we carried out two-dimensional electrophoresis according to the NEPHGE protocol. Fig. 3B shows the separation pattern of the two topoisomerase variants from normal liver. In the first dimension, the two species migrated as several forms to a pH between approximately 8.2-8.6. The isoelectric point (PI) of the two variants is therefore around these values. The same result was obtained with a regenerating liver extract (data not shown). After protein dephosphorylation by alkaline phosphatase, each variant migrated as a single form at pH 8.6 (Fig 3C). In correlation with this, the DNA relaxation of the extract was abolished (data not shown).

362 A

B PH 8 . 6 - 8 . 2 OH-

c first

C

dimension

L

+first

dimension

pH8.6

H+

I

OH-

I

H+

I

71’

44

-

Fig. 3. Separation of the two topoisomerase4 variants from normal liver by two-dimensional gel electrophoresis. Topoisomerase I was detected by Western blot with antiserum directed against a cloned fragment of human topoisomerase I. The directions of the first (NEPHGE) and second (SDS/PAGE) dimensions are indicated (B and C). The normal-liver extract was also separated only by SDS/PAGE, run in parallel (A). (A and B) Normal liver extract. (C) The same extract dephosphorylated by alkaline phosphatase. The small arrow indicates the origin of the anodic end of the NEPHGE gels.

Differential affinity of topoisomerase I for chromatin during liver regeneration

Salt elution of topoisomerase I f r o m normal liver is correlated with its activity

Soluble and matrix fractions obtained from nuclei treated with 5-250 mM NaCl as well as total nuclear extract, obtained after treatment of nuclei with 1 M NaC1, and cytoplasmic extract (see Experimental Procedures) were analysed by Western blotting and assayed for DNA-relaxation activity. Fig. 4 shows that topoisomerase-I activity was correlated with the presence of the two polypeptides which reacted with anti(topoisomerase I) serum. Elution of topoisomerase I from chromatin became more efficient as NaCl concentration increased from 5 mM to 250 mM. The fraction of topoisomerase-I antigen remaining attached to the nuclear matrix decreased in parallel. Approximately 50% of topoisomerase I was eluted at 250 mM NaC1. No topoisomerase-I antigen was detected in the cytoplasmic fraction. Differential elution of topoisomerase I from regenerating-liver nuclei

The salt-elution profile of topoisomerase I was determined in regenerating-liver extracts by Western blot. As shown in Fig. 5 , nearly 10O0/~of the enzyme was eluted from 16-hregenerating-liver and 24-h-regenerating-liver nuclei at 250 mM NaCI.

DISCUSSION In a previous study (Sobczak et al., 1989), we showed that topoisomerase-I-encoding mRNA was accumulated during

cell proliferation in vivo in regenerating rat liver. The purpose of the present study was to answer the following questions. Does the amount of topoisomerase I polypeptide(s) increase in parallel with the mRNA during liver regeneration? Are the enzymic properties of topoisomerase I modified during this process, in relation to possible post-translational modifications? Is the association of topoisomerase 1 with chromatin modified during liver regeneration? The data presented in this paper indicate that, after hepatectomy, the amount of topoisomerase I in each nucleus does not increase significantly. In contrast, topoisomerase-1mRNA level is found increased 2.5 - 5-fold over normal during the period of time from 16-30 h post-hepatectomy (Sobczak et al., 1989). Together, these results suggest that topoisomerase I is less stable in proliferating cells than in quiescent cells in the liver. Another hypothesis is that topoisomerase-I mRNA remains untranslated during this period of time. In normal as well as in regenerating liver, we have observed two variants with apparent molecuiar mass of about 97 kDa and 100 kDa which react with anti-(topoisomerase I) sera. Topoisomerase I was previously purified from rat liver with a reported molecular mass of approximately 67 kDa (Champoux and McConaughy, 1976), presumably produced by proteolysis of a 100-kDa native form, as suggested for the enzyme from HeLa cells and calf thymus by Liu and Miller (1981) and Schmitt et al. (1984). Similar existence of two polypeptides of 98 kDa and 102 kDa was only reported in cultured mouse mammary carcinoma cells (Ishii et al., 1983). The authors suggested that the two forms were structurally related but could differ by an unidentified post-translational modification; both forms seem to possess the topoisomeraseI activity. In human placenta, two chromatographic forms of

363

A

1

2

3

4

t

t

t

t

5

6

7

8

9

1

0

195+

105+ 71-

44 +

B

t

t

41

Fig. 4. Correlation between DNA-relaxation activity in normal-liver extracts and solubilisation of topoisomerase I by salt treatment. (A) Western blot incubated with the antiserum directed against a cloned fragment of human topoisomerase I. Proteins from 5 x lo6 nuclei were loaded in each lane. Lane 1 , total nuclear extract obtained after incubation ofnucleiin buffer B containing 1 M NaC1; lane 2, cytoplasmic extract; lanes 3 -6, soluble nuclear proteins obtained after incubation of nuclei in buffer B containing respectively 5 (lane 3), 50 (lane 4), 125 (lane 5) and 250 (lane 6) mM NaC1. Lanes 7-10, corresponding matrix nuclear proteins in 5 (lane 7), 50 (lane S), 125 (lane 9) and 250 (lane 10) mM NaCl. (B) Relaxation assays were performed in the presence of 250 mM KCI with the proteins extracted from 750 nuclei. I, supercoiled DNA; Ir, fully or partly relaxed DNA. Molecular masses, in kDa, are indicated on the left.

Similar to the protein level, the activity of topoisomerase I in each nucleus or relative to total nuclear proteins does not vary significantly during the cell cycle. Our results are in agreement with those previously obtained in regenerating liver by Champoux et al. (1978) and Duguet et al. (1983). Contradictory results have been obtained in other experimental systems. For example, a considerable (40-fold) increase of enzymic activity was observed during the S phase in synchronized human lymphocytes when compared to the stationary phase (Rosenberg et al., 1976). In contrast, a 25-fold increase was measured in S phase in cells stimulated for proliferation (Tdudou et al., 1984; Tricoli et al., 1985; Romig and Richter, 1990). Magnesium stimulation and sensitivity to ATP of topoisomerase-I activity were identical in normal and regenerating liver. A similar effect of Mg2+ on DNA relaxation has been reported in other cell types (Coderoni et al., 1990; Goto et al., 1984; Der Garabedian et al., 1991). In standard experimental conditions (virtually no Mg2+), ATP does inhibit DNA relaxation mediated by liver topoisomerase I as previously described in various cell types (Low and Holden, 1985; Castora and Kelly, 1986; Chen and Castora, 1988). However, in our study, this inhibition is suppressed at high magnesium concentrations (20 mM). It is possible that ATP inhibits relaxation by competing (mainly through ionic interactions) with the DNA substrate for topoisomerase binding. Magnesium would then trap ATP, lowering its binding to topoisomerase and thus reducing its competitive effect with DNA. Alternatively, the relaxation activity observed may be due to type-I1 topoisomerase which requires Mg2 and ATP. This possibility is however, unlikely, since the effect of Mg2+ is the same in extracts of normal and regenerating liver although topoisomerase I1 is considerably increased in regenerating liver (Duguet et al., 1983). After dephosphorylation, we observed a decrease of heterogeneity of the p l of the two apparent-molecular-mass variants indicating that both variants of the native enzyme contained a non-phosphorylated precursor together with others forms of variable degrees of phosphorylation. Moreover, our study does not show difference between the phosphorylation state of topoisomerase I in normal and regenerating liver. In both situations, the DNA relaxation was decreased by treatment with phosphatase. This is in agreement with results observed in Novikoff rat hepatoma (Durban et al., 1985), in Xenopus ovaries (Kaiserman et al., 1988) and in Chinese hamster cell line (Pommier et al., 1990). Finally, topoisomerase I appeared to be tightly bound to chromatin as previously observed in regenerating liver (Nishizawa et al., 1984). We observe that the salt-elution profile of topoisomerase I depends on the cell cycle. Nearly 100% of the enzyme is eluted from nuclei at 250 mM NaCl during GljS and S phase (respectively 16 h and 24 h after hepatectomy). In contrast, 50% of the enzyme remains bound to chromatin in quiescent cells (normal liver). Our results are conflicting with those previously published by McConaughy et al., (1981) who found that 70% of topoisomerase I remained bound to chromatin from growing 3T3 mouse fibroblasts. The reason for this discrepancy is unknown. Hence, although the in-vitro activity of topoisomerase I appeared identical in normal and regenerating liver, this activity may be regulated in vivo by its association with chromatin and by post-translational modifications that are no detectable in our experimental conditions. Purification and separation of the two topoisomerase-I variants should facilitate further studies. +

A

195

-

1

B 2

3

4

5

6

1

2

3

4

5

6

10571

44

c

Fig. 5. Salt elution of topoisomerase-I variants from regenerating-liver nuclei determined by Western blot with the antiserum directed against a cloned fragment of human topoisomerase I. Proteins from 5 x lo6 nuclei were loaded in each lane. (A) 16 h post-hepactomy; (B) 24 h post-hepatectomy. Lanes 1 - 3, soluble nuclear proteins obtained after incubation in buffer B containing respectively 5 (lane I), 125 (lane 2) and 250 (lane 3) mM NaCI. Lanes 4-6, corresponding matrix proteins in 5 (lane 4), 125 (lane 5) and 250 (lane 6) mM NaCl. Molecular masses, in kDa, are indicated on the left.

topoisomerase I have been characterized but with no differences in molecular mass (100 kDa; Holden et al., 1990). In our case, the two topoisomerase-I variants are antigenically related but we were unable to separate them. We therefore do not know whether both variants possess the topoisomerase-I activity. The two rat-liver variants differ only in molecular mass and show the same behaviour in a two-dimensional NEPHGE. They have the same pZ of 8.2 - 8.6, comparable to the value found in calf thymus (8.1 -8.3; Schmitt et al., 1984) and in Novikoff hepatoma cells (8.4; Durban et al., 1983).

We are grateful to Dr Richter for the generous gift of anti(topoisomerase-I) sera without which a part of this work would not have been possible, to Dr M. Nadal for helpful assistance concerning the computer analysis, to many members of the Laboratory Enzyrnologie des acides nucltiques and to Dr G. Mirambeau for comments on the manuscript. This work was supported by a grant from the Association pour la Recherche contre le Cancer and by the Centre National de la Recherche Scientifique (Unit6 associke n “554).

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Comparison of biochemical properties of DNA-topoisomerase I from normal and regenerating liver.

Biochemical properties of topoisomerase I from normal and regenerating rat liver were analysed using crude or fractionated nuclear extracts. We could ...
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