DEVELOPMENTAL
BIOLOGY
146,4-11 (1991)
The 165kDa DNA Topoisomerase I from Xenopus Levis Oocytes Is a Tissue-Specific Variant ROBERTE.RICHARDANDDANIEL F. BOGENHAGEN Department of Pharmacological Sciences, State University of New York, Stony Brook, New York 11794 Accepted March 6, I!%1 Two forms of topoisomerase I can be purified from Xenopus 2aevi.s. A protein with a molecular mass of 165 kDa has been identified as topoisomerase I in ovaries (Richard and Bogenhagen, 1989. J. Biol. Chem. 264,4704-4709). When a similar purification is performed using liver tissue, topoisomerase I is purified as a DO-kDa protein. Separate rabbit antisera were raised against oocyte and liver topoisomerase I polypeptides. Each antiserum reacts in immunoblottingor immunoprecipitation procedures only with the tissue-specific topoisomerase I polypeptide against which it was generated. The failure of the antiserum raised against liver topoisomerase I to cross-react with the oocyte enzyme suggests that the smaller topoisomerase I is not derived from the 165-kDa oocyte enzyme by proteolysis. X laevis tissue culture cells lysed and processed in the presence of SDS contain the IlO-kDa form of topoisomerase I. The 165-kDa form of topoisomerase I disappears during oocyte maturation in vitro. @ 1991 Academic PRWS, IX.
We recently purified a topoisomerase I with an unusually large molecular mass of 165 kDa from the ovaries of DNA topoisomerases catalyze changes in DNA topol- X Zaevis (Richard and Bogenhagen, 1989). Prior to this ogy by cleaving and resealing the phosphodiester back- finding, the largest topoisomerase I described had been bone of the DNA molecule (Wang, 1985). In eukaryotes, the 135-kDa enzyme of Drosophila melanogaster (Javathere are two major types of topoisomerases, I and II. herian et al, 1982). We assumed that this difference in The cDNAs of three eukaryotic type I enzymes have molecular weight compared to other purified type I been cloned and sequenced. These include human topoisomerases was due to species-specific variability. (D’Arpa et al, 1988), Saccharomyces cerevisae (Thrash et In order to test this hypothesis, we decided to examine al., 1985), and Schixosaccharomyces pombe (Uemura et other tissues from X Zaevis. Our initial purification of aZ., 1987). The human gene is similar in size and se- topoisomerase I from liver of adult X laevis indicated quence to the two yeast genes. Human topoisomerase I that the 165-kDa ovarian enzyme was not present. appears to be a single copy gene that maps to chromo- Rather, a more conventionally sized type I topoisomersome 20 and encodes a protein of 765 amino acids (Juan ase llO-kDa polypeptide was purified. In this paper we et ab, 1988). show that the llO-kDa form is representative of the preTopoisomerase I is involved in a number of cellular dominant, somatic form of topoisomerase I and is not processes including transcription (Zhang et al., 1988; derived from the 165-kDa topoisomerase I by proteolyBrill and Sternglanz, 1988; Stewart et ah, 1990), replica- sis. The 165-kDa form is restricted to oocytes and disaption (Brill et al., 1987;Kim and Wang, 1989a), and recom- pears during oocyte maturation. bination (Christman et al., 1989; Kim and Wang, 198913). The cell must be able to selectively recruit and modulate MATERIALS AND METHODS topoisomerase activity in each of these processes. Recent observations of topoisomerase heterogeneity sug- Materials gest that modulation of topoisomerase activity may ocPhosphocellulose was purchased from Whatman cur by expression of different forms of topoisomerase (Chung et al., 1989; Wallis et aZ.,1989). Since most stud- (Clifton, NJ); immobilon was from Millipore (Bedford, ies have been performed in yeast and mammalian cell MA); hydroxyapatite (HA) Ultrogel was from IBF; culture systems, little is known about topoisomerase I phenyl-Superose HR lO/lO and Protein A-Sepharose during the development of a higher eukaryote. Xenopus were from Pharmacia LKB Biotechnology (Piscataway, laevis offers the advantages of a well-characterized de- NJ); agarose was from Bethesda Research Laboratories velopmental system, in which biochemical amounts of (Bethesda, MD); BCIP/NBT phosphatase substrate system was from Kirkegaard and Perry Laboratories enzymes can be obtained. INTRODUCTION
001%1606/91 $3.00 Copyright All rights
(c) 1991 by Academic Press. Inc. of reproduction in any form reserved.
4
RICHARD AND BOGENHAGEN
X laevis Topoisomerase
(Gaithersburg, MD); PMSF,l benzamidine-HCl, and prestained protein molecular weight markers were from Sigma (St. Louis, MO). The X Zaevis cell line, A6, was obtained from American Type Culture Collection and propagated in 0.75X NCTC 135 media containing 10% fetal bovine serum. Freund’s complete and incomplete adjuvant were from DIFCO (Detroit, MI). Topoisomerase Assay The assay for topoisomerase I activity and the method to radiolabel topoisomerase I were performed as published (Richard and Bogenhagen, 1989). Purification
of Liver Topoisomerase I
The preparation of a nuclear extract from liver employed a variation of the method of Dignam et al. (1983). All operations were carried out at 4°C. Livers were removed from matureX laevis, washed and minced in PBS with 2 mM DTT, 0.4 mM PMSF, 1 mM benzamidineHCl. The tissue was placed in 4 vol of a hypotonic solution consisting of 10 mM Hepes, pH 7.9, 1.5 mM MgCl,, 10 mM KCl, 1 mM DTT, 0.4 mM PMSF, 1 mM benzamidine-HCl, 2.5 pg/ml aprotinin, 2.5 pg/ml leupeptin for 15 min and homogenized with a motor driven glassTeflon homogenizer. Nuclei were pelleted by spinning at 2000 rpm in a Beckman JA-14 centrifuge. The supernatant was discarded. The nuclear pellet was homogenized and resuspended in 5 vol of 20 mM Hepes, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl,, 0.2 mM EDTA, 0.4 mM PMSF, 1 mM DTT, 1 mM benzamidine-HCl, 2.5 pg/ml aprotinin, 2.5 pg/ml leupeptin and spun in a Beckman Ti 70 rotor at 40k rpm for 1 hr. The supernatant (nuclear extract SlOO) was frozen and stored at -80°C (fraction I). The frozen extract was diluted with Buffer A (20 mM Tris-HCl, pH 7.5, 0.2 mM EDTA, 15% glycerol, 1 mM DTT, 1 mM benzamidine-HCl, 0.4 mM PMSF, 1 pg/ml leupeptin, 1 pg/ml aprotinin) to a NaCl concentration of 0.2 M. The protease inhibitors at the indicated concentrations were included in all subsequent buffers. The diluted extract was loaded on a phosphocellulose column (2.5 X 15-cm) and washed with 0.5 MKCl in Buffer A. A 1.2 M KC1 step with Buffer A lacking EDTA was performed to elute topoisomerase I activity (fraction II). This high salt step was loaded directly on a 5 ml HA Ultrogel column (1.6 x 4-cm) equilibrated in Buffer A without EDTA. Topoisomerase I activity was step ’ Abbreviations used: SDS, sodium dodecyl sulfate; PMSF, phenylmethanesulfonyl fluoride; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; Hepes, N-hydroxyethylpiperizine-W-2ethanesulfonic acid; EDTA, ethylenediaminetetraacetic acid; PBS, phosphate-buffered saline; TCA, trichloroacetic acid.
I Is Tissue Specific
5
eluted with 0.8 M KP,, pH 6.9 (fraction III). The step from the hydroxyapatite column was adjusted with 3 M (NH&SO, to a final conductivity equivalent to that of 1.3 M (NH&SO,. This was filtered and loaded on the fast protein liquid chromatography column phenyl-Superose HR lO/lO and eluted with a gradient of (NH&SO, decreasing from 1.3 to 0.1 M. Topoisomerase I activity eluted at approximately 0.8 M (NH&SO, (fraction IV). Generation of Polyclonal Antisera Methods were similar to those described in Harlow and Lane (1988). New Zealand White rabbits were used to generate polyclonal antisera against the 165-kDa ovarian topo I and the llO-kDa liver topo I. Fraction IV from the liver purification was used to gel purify the llO-kDa form. The SDS-gel filtration fraction described by Richard and Bogenhagen (1989) was used to gel purify the 165-kDa form. Proteins were run on SDSPAGE (Laemmli, 1970), identified by copper staining (Lee et al., 1987), excised from the gel, and homogenized in PBS/50% complete Freund’s adjuvant (for the initial injection, subsequent injections were performed with incomplete Freund’s adjuvant). The popliteal lymph nodes were the sites of all injections. Between 5 and 15 pg of protein was used for each injection. Anti-llO-kDa antisera was routinely used at a dilution of l/2000. Anti-165-kDa antisera was used at a dilution of l/10000. When affinity-purified antiserum was required, the method of Smith and Fisher (1984) was employed with minor changes. Immobilon membranes were substituted for nitrocellulose and the NH,SCN washes were omitted. Antibody directed against the 165-kDa polypeptide was affinity-purified from a preparative blot of ovarian fraction III protein. Antibody against the llOkDa form was affinity-purified from a preparative immunoblot of A6 tissue culture cell extract chromatographed on an SDS-hydroxyapatite column (see below). Immunoprecipitation
of Topoisomerase I Activity
Protein A-Sepharose was loaded with IgG by incubating the resin on a rotator with an equal volume of antiserum for 2 hr at 4”C, removing the antiserum, and repeating the procedure. The beads were washed with IP Buffer (20 mM Tris, pH 7.6,150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 0.4 mM PMSF, 1 mM benzamidineHCl) and stored in IP Buffer with 0.02% NaN, at 4°C. Immunoprecipitations were carried out in IP Buffer on a rotator, the beads spun out, and the supernatant was assayed for topoisomerase I activity.
6 Denaturing
DEVELOPMENTAL BIOLOGY
TABLE 1 PURIFICATION OF TYPE I TOPOISOMERASE FROM X laevis LIVER
Lysis of X laevis A6 Tissue Culture Cells
Cells grown on monolayer were removed from T-150 tissue culture flasks (~10’ cells) with versene, pelleted, resuspended in lysis buffer (2% SDS, 50 mM Tris-HCl, pH 7.6, 10 mM DTT), and boiled for 10 min. DNA was sheared by passing the sample through a 26-gauge needle. The extract was spun at 14,000gfor 10 min, and the supernatant removed, frozen in liquid N,, and stored at -80°C. When used, the extract was thawed, diluted in dilution buffer (50 mM Tris-HCl, pH 7.5,100 mM NaCl, 0.2% SDS, 1 mM benzamidine-HCl, 0.4 mMPMSF), and loaded on a l-ml HA Ultrogel column equilibrated in dilution buffer. The column was washed in 0.1 M NaPi, pH 6.9, and stepped with 0.5 M NaPi, pH 6.9. Fractions from this column were TCA precipitated, separated on 6% SDS-PAGE, transferred to immobilon membranes, and probed with either affinity-purified antibody. Transfer of proteins to immunoblots was performed using passive or electroblot transfer conditions as described in Harlow and Lane (1988).
VOLUME 146,199l
Volume (ml)
Fraction I. II. III. IV.
NE-S100 Phosphocellulose Hydroxyapatite Phenyl-Superose
130
50 9 6
Protein (w) 1070 17.5 9.9 0.1
Activity (units)
Sp Act (U/mg)
Yield (7%)
800,000 500,000 360,000 200,000
750 28500 36000 2 x lo6
100 62.5 45
25
trotransfer, the immobilon membrane was probed with affinity-purified anti-165-kDa antibody. RESULTS
Purification
of Topoisomerase I from Liver
The procedure used to purify topoisomerase I from the liver nuclear extract was essentially the same as that previously used to purify topoisomerase I from ovaries (Table 1). The first chromatographic step, involving stepwise elution from phosphocellulose, provided a 35fold purification with good recovery of activity, but left Immunoprecipitations of Topoisomerase I from the enzyme in a relatively large volume of high salt Detergent-Treated Oocytes buffer. The subsequent hydroxyapatite column was used Stage VI oocytes were collected after treatment with largely to concentrate the enzyme. Step elution was subcollagenase as described in Luke and Bogenhagen stituted for gradient elution from the hydroxyapatite (1989). Maturation was induced by addition of proges- column since gradient elution gave little additional puriterone to a final concentration of 5 pg/ml. At each time fication. The enzyme eluted from hydroxyapatite was point, 100 oocytes were removed to a 1.7-ml microfuge applied to a phenyl-Superose column and eluted with tube and 0.5 vol of extraction buffer (80 mM P-glycerol- solutions of decreasing (NH&SO, concentration. The phosphate, 20 mMEGTA, 15 mMMgCl,, 0.1 MNaCl, 0.2 specific activity of fraction IV is in the range of purified mM PMSF, 2 mM DTT, and 1 pg/ml leupeptin) was type I topoisomerases from other sources (Tricoli and added. The oocytes were then spun at the maximal set- Kowalski, 1983; Martin et al., 1983). This purification ting of a microcentrifuge, 16,000 RCF, for 15 min at 4°C. procedure was designed so that the three columns could The clear supernatant (S16) was removed, mixed with be run quickly, in approximately 7 hr, with minimal hanan equal amount of a solution containing 2% SDS, 10 dling of the eluted fractions. Time consuming topoisomerase I assays were carried out only once, after the mMDTT, 0.2 mMPMSF, 2.5 mMEDTA, 2 mMbenzamidine-HCl, 1 pg/ml leupeptin, and boiled for 5 min. The third chromatographic step. The fraction IV enzyme 165-kDa topoisomerase I present in the SDS denatured was stored frozen as a (NH,),SO, precipitate while the S16 was immunoprecipitated following addition of 3 vol enzyme assay data were collected. A wide range of proof a Triton Buffer (4% Triton X-100, 150 mM NaCl, 50 tease inhibitors were included in all of the buffers. The mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 1 mM benzami- polypeptide pattern on a polyacrylamide gel and topoisomerase I enzyme assays of the phenyl-Superose fracdine-HCl, 0.4 mM PMSF, 1 pg/ml leupeptin) essentially as described in Luke and Bogenhagen (1989). Twenty tions are shown in Fig. 1. The topoisomerase activity microliters of Protein A-Sepharose beads loaded with eluted with the first proteins to elute from the column. the anti-165-kDa enzyme was used for each of the im- The majority of the proteins that adsorbed to the colmunoprecipitations. After washing the beads, 25 ~1 of a umn eluted after fraction 12 and are not shown in Fig. 1. solution loading buffer (4% SDS, 20 mMDTT, 10% glyc- One major polypeptide with a molecular weight of erol, 120 mM Tris-HCl, pH 8.8, with bromophenol blue 110,000 and a second polypeptide with a molecular as a mobility marker) was added. The mixture was weight of 80,000 copurified with the activity (Fig. lA, boiled for 2 min and treated with 0.1 M iodoacetamide. arrowhead). Experiments presented below suggest that The beads were sedimented and the supernatant was this 80-kDa polypeptide is a breakdown product derived loaded onto a SDS-polyacrylamide gel. Following elec- from the IlO-kDa polypeptide. This enzyme is clearly a
RICHARD
A
AND BOGENHAGEN
L 1 2 3 4 5 6 7 6 9 10 1112
160D 116 P 64 D
(Fig. 2). This control demonstrated that the ovary enzyme has the high molecular weight reported previously. Never in the course of our experiments has a 165-kDa polypeptide radiolabeled or coeluted with topoisomerase activity in a fraction of liver enzyme.
46 r=-
Antisera Against the Ovarian Topoisomerases
FIG. 1. Phenyl-Superose HR 1000 chromatography of topoisomerase I from liver. (NH&SO, was added to the enzyme fraction eluted from hydroxyapatite (fraction III) until a final conductivity equivalent to that of 1.3 M (NH&SO, was obtained. The solution was then chromatographed on a phenyl-Superose HR lO/lO column as described under Materials and Methods (A) One hundred-microliter samples of the l-ml fractions eluting from the column were TCA precipitated and treated as described by Laemmli (1970). The samples were then subjected to electrophoresis on 6% SDS-polyacrylamide gels. Silver staining was performed as described by Wray et ul. (1981) after Coomassie staining and destaining. Lane L, 100 ~1 of fraction III. (B) Type I topoisomerase assays of the fractions eluting from the phenyl-Superose HR 10110 column. One microliter of l/20 diluted samples from the marked fractions was assayed in a standard reaction. Lane +, 1 ~1 of similarly diluted load (fraction III) was assayed.
type I topoisomerase. Its activity is independent of ATP, it is unable to decatenate kinetoplast DNA, and it has the same sensitivity to camptothecin as the oocyte topoisomerase I described previously (Richard and Bogenhagen, 1989; data not shown). We employed the method of radiolabeling topoisomerase I as outlined previously to identify the polypeptides capable of covalently binding to DNA (Richard and Bogenhagen, 1989). This method specifically labels topoisomerases (Chow and Pearson, 1985). Liver topoisomerase I purified only through fraction III was used in this experiment in order to provide a sufficiently concentrated preparation at the earliest possible purification step, thereby minimizing the possibility of degradation during additional purification. Proteins were denatured by addition of SDS, renatured, incubated with radiolabeled DNA, and then digested with DNAase I (Fig. 2). Only two proteins were labeled by this procedure despite the complex set of polypeptides present in fraction III (see Fig. lA, lane “L,” for polypeptide composition). These DNA-labeled polypeptides have the same molecular weights as the two polypeptides that coeluted with topoisomerase I activity in Fig. 1. Radiolabeling of liver fraction IV proteins resulted in the same pattern (data not shown). As a control, fraction III from an ovarian preparation (Richard and Bogenhagen, 1989) was treated similarly and run on the same gel
and Liver Type I
The purifications mentioned above and in our previous report produced sufficient quantities of gelpurified liver and ovarian topoisomerase I to generate specific polyclonal antisera in rabbits. These two antisera were then used to study the possibility of shared antigenic determinants between the two polypeptides and their respective breakdown products. On protein blots, each antiserum recognized the form of topoisomerase I against which it was raised (Fig. 3), but failed to cross-react with topoisomerase I from the other source. Although neither antiserum neutralizes enzyme activity, each antiserum was capable of immunoprecipitating enzyme activity from the appropriate tissue, as shown in Fig. 4. In agreement with the immunoblotting results, the antisera were not able to immunoprecipitate activity from extracts derived from the other source tissue, i.e., anti-ovarian topoisomerase I antibody did not recognize liver activity and vice versa. These results indicate
Liver
Ovary
180D
116D 84
b
48
L>
of DNA topoisomerase I. The radioFIG. 2. Active site radiolabeling labeling method described under Materials and Methods was used to identify the molecular weight of the type I topoisomerase in fraction III. A. A 50-4 sample of fraction III, processed to radiolabel topoisomerase I, was TCA precipitated, fractionated on 6Y0 SDS-polyacrylamide gel, and subjected to autoradiography. (B) An 80-J sample of fraction III from an ovarian preparation (Richard and Bogenhagen, 1989) was treated as in A. In both lanes the dark band at the bottom represents degraded DNA at the dye front.
8
DEVELOPMENTAL BIOLOGY
VOLUME 146. 1991
ovarian extracts (data not shown). In Fig. 5B, a duplicate immunoblot probed with the affinity-purified anti165kDa antibodies revealed no specific staining. This analysis confirms that tissue culture cells contain the llO-kDa form of topoisomerase I. We estimate that if the 165-kDa polypeptide were present in liver and/or tissue culture cells at a level > 5% of the llO-kDa form it would have been identified by the methods we employed. The 165kDa Form of Topoisomerase I Disappears upon Oocyte Maturation
FIG. 3. Immunoblotting of the 165. and IlO-kDa forms of topoisomerase I with antisera raised against each form. Liver extract (75 ~1 of fraction III) and ovarian extract (200 11 of fraction III) were TCA precipitated, divided equally into two lanes on a 6% SDS-polyacrylamide gel, subjected to electrophoresis, and electroblotted to an immobilon membrane. Lanes marked 1 contain liver extract, lanes marked 2 contain ovarian extract. (A) The immunoblot was probed with antisera raised against the 165.kDa type I topoisomerase. (B) The immunoblot was probed with the antisera raised against the DOkDa form.
that these polyclonal antisera recognize distinct epitopes not shared by the two forms of topoisomerase I.
Since we were not able to detect the 165-kDa topoisomerase I in somatic cells, it appears to be an oocytespecific form of the enzyme that is lost during early development. The Xenopus system provides an opportunity to follow the fate of stored oocyte proteins. We prepared extracts from stage VI oocytes after they had been incubated either in the absence or in the presence of progesterone to induce oocyte maturation. Oocyte maturation results in germinal vesicle breakdown and development of the oocyte into an unfertilized egg. These extracts were processed to immunoprecipitate the 165-kDa form of topoisomerase I, and the protein was detected by immunoblotting. As shown in Fig. 6, the disappearance of the 165-kDa topoisomerase I coincides with oocyte maturation. We have not been able to detect A
anti-110 Enzyme Preim. Immune
-
-
Liver 20 -
40 -
10
20
* 40
ovary 20 -
40 10 -
20
40
-
-
Liver 20 -
40 -
10
20
40
o-=-Y 20 -
40 -
10
20
40
X laevis Tissue Culture Cells Contain the IlO-kDa Topoisomerase I
The possibility existed that the 165kDa form was present in liver but could not be isolated intact. We believed this not to be the case since we observed no crossreactivity between the antibodies directed against the liver and oocyte topoisomerases. Nevertheless, we decided to analyze topoisomerase I present in X laevis tissue culture cells as another somatic cell type. Proteolysis in these cells should be significantly less than in liver preparations. To limit proteolysis, the cells were lysed in the presence of 2% SDS, and the enzyme was concentrated by chromatography on hydroxyapatite in the presence of SDS and subjected to SDS-PAGE. Immunoblots were probed with both antisera following affinity purification. This experiment showed that only the llO-kDa form of topoisomerase I is present in X laevis tissue culture cells (Fig. 5A). The absence of the 80-kDa form suggests that this method of purification successfully limited degradation. The elution conditions employed effectively concentrate the 165-kDa form from
B
anti-185 Enzyme Preim. I-une
FIG. 4. Immunoprecipitation of topoisomerase I activity from liver and ovary fractions. Twenty units of topoisomerase I from fraction III of a liver preparation or from fraction III of an ovarian preparation was incubated with IgG fractions bound to Protein A-Sepharose beads. Ten, twenty, or forty microliters of beads was added to the fractions as marked. One-twentieth of the immunoprecipitation supernatant was added to the topoisomerase I reactions shown in this figure. (A) Immunoprecipitations performed with the anti-llO-kDa antisera with either liver or ovarian activity added as marked. (B) Immunoprecipitations performed with anti-165kDa antisera. (Preim. stands for added preimmune serum).
X lneris Topoisowuvuse I Is Tissue Specilc
RICHARDANDBOGENHAGEN
A
B LFt
w
0.5 $?
L
Ft
W
e 0.5
$
160 11684r 58 489
FIG. 5. X. Irter.is tissue culture cells contain the IlO-kDa somatic form of topoisomerase I. X lof~c+s tissue culture cells were processed as under Materials and Methods, and fractions from the SDS-hydroxyapatite column were separated on a 6%1 SDS-polyacrylamide gel. The samples were transferred to immohilon membranes and probed with either the anti-llO-kDa (A) or anti-165-kDa (B) antisera following affinity purification as described under Materials and Methods. Lane L contains the column load; lane Ft contains the flow through; and lane W contains the column wash. The lane marked “Liver” contains 50 ~1 of fraction III of a liver preparation; the lane marked “Ovary” contains 25 ~1 of fraction III of an ovarian prcparation. Approximately 1/20th of the total 0.5 MNaP, step was loaded in lane 0.3 (lo7 cells worth of topoisomerase I). Similar amounts of protein were added in each lane as determined by staining with Coomassir blue (-1/200th of total load and flow through are loaded lane L and lane Ft). The 80-kDa breakdown product found in the liver prcparation is marked with an asterisk (*).
the llO-kDa form of topoisomerase I in unfertilized eggs either using immunoprecipitation as in Fig. 6 or using a chromatographic technique similar to that used for cultured cells in Fig. 5 (data not shown). These results indicate that the 165-kDa oocyte-specific topoisomerase I is not a storage form of the enzyme for use in embryonic development.
9
than 165 kDa and possess topoisomerase I activity (Richard and Bogenhagen, 1989). These polypeptides are recognized by the anti-165kDa antiserum and do not comigrate with the somatic enzyme on SDS-PAGE (unpublished observations). We believe that the smaller polypeptides described in our previous paper are degradation products of the 165-kDa oocyte form. We have considered the possibility that the discrepancy in molecular weight between ovarian and liver topoisomerase I was due to extensive proteolysis of the 165-kDa form in liver homogenates. Topoisomerase I is subject to proteolysis when purified from liver (Martin et al., 1983). Nevertheless, we consider that the molecu-
116 *
B
I
40T
1
20 -c
/ /
0
DISCIJSSION
Recently, we published the purification of a high molecular weight topoisomerase I from X Zaevis ovaries (Richard and Bogenhagen, 1989). In this paper, we used a similar purification protocol to identify a type I topoisomerase with a molecular weight of 110 kDa from liver. To our knowledge, this work represents the first report of a tissue-specific variation of topoisomerase I. Chung et nl. (1989) recently described a second variant of type II topoisomerase in human cells. Whether these enzymes are differentially expressed in different tissues has not been established. Attardi ef (xl. (1981) and Kaiserman ef al. (1988) have purified forms of topoisomerase I with molecular weights of approximately 67 and 110 kDa, respectively, from X 1ac~i.s ovaries. We have also described polypeptides derived from ovarian extracts that are smaller
i
e-0 0
3
Hours
’
g
FIG. 6. The 165-kDa form of topoisomerase I is lost during oocyte maturation. (A) Immunoblot of topoisomerase I immunoprecipitated with the anti-l65-kDa antiserum. One thousand stage VI oocytes were collected after collagenase treatment of ovarian tissue as described (Luke and Bogenhagen, 1989). Half of the sample was treated with progesterone (lanes labeled +) while the remaining half served as the untreated control (lanes labeled -). One hundred oocytes were removed after 0,3,6 or 9 hr of incubation, and were processed to an S16 extract as described under Materials and Methods and by Luke and Bogenhayen (submitted). Topoisomerase I was immunoprecipitated with the anti-165.kDa antiserum and detected by SDS-PAGE and immunoblotting as described under Materials and Methods. Lane M contains mobility markers of prestained cu,-macroglobulin (180 kDa) and @-galactosidase (116 kDa). Lane C contains a positive control of 500 units of ovarian topoisomerase I purified through step III as described in Richard and Bogenhagen (1989). Lanes labeled “0” contain samples immunoprccipitated with preimmune or anti-165.kDa antiserum from freshly isolated oocytes. (B) Oocytes were scored for the percentage exhibiting white spots as a function of the time of i?r c,itro incubation. White spot formation is a marker of germinal vesicle breakdown and oocyte maturation.
10
DEVELOPMENTALBIOLOGY
lar weight difference between the oocyte and the somatic type I topoisomerases reflects a real difference in the form of the enzyme for three reasons. First, particular care was taken to complete the purification in the shortest possible time in the presence of a wide variety of protease inhibitors. Labeling of the liver enzyme with DNA showed no evidence of 165-kDa topoisomerase I from this source (Fig. 2). Second, the lack of cross-reactivity between the two antisera suggested that the llOkDa form is not a degradation product, but rather an alternate form of the enzyme (Figs. 3 and 4). The fact that the anti-165-kDa antiserum does not recognize the liver form of topoisomerase I might not be noteworthy if the major epitopes of the protein recognized by the antiserum were in a domain removed by proteolysis. However, this sort of argument cannot explain the inability of the anti-llO-kDa antiserum to recognize the oocyte form of topoisomerase I. Finally, we also analyzed the size of topoisomerase I from another somatic cell type not known for proteolysis, X Zaevis tissue culture cells. Extracts produced from tissue culture cells lysed in the presence of SDS contained only the llO-kDa form of the enzyme (Fig. 5). A similar immunoblot probed with the anti-165-kDa antisera showed no specific bands. What is the Relationship between Oocyte and Somatic Forms of Topoisomerase I? The large difference in molecular weight between the oocyte and the somatic forms of topoisomerase I is unlikely to result from differential post-translational modification. The 165-kDa form of topoisomerase I is a phosphoprotein, but complete removal of phosphates by potato acid phosphatase produces only a slight increase in gel mobility (R.E.R., unpublished observation). The 165-kDa form of the enzyme does not appear to be modified by 0-glycosylation (R.E.R., unpublished observation; Holt and Hart, 1986). It seems most likely that these two forms of the enzyme are derived from different mRNAs. There may be two differentially regulated genes for topoisomerase I in the X. laevis genome. Alternatively, the two forms of enzyme might result from tissue-specific differences in the splicing of a primary transcript. Experiments to characterize cDNAs for these two forms of topoisomerase I are in progress. Possible Roles for the Oocyte-SpeciJic X5-kDa Topoisomerase I The oocyte-specific nature of the 165kDa form of topoisomerase I is further emphasized by the observation that this form of the enzyme is specifically degraded during oocyte maturation (Fig. 6). This situation presents an interesting contrast to the observation that the quantity of type II topoisomerase increases nearly
VOLUME146. 1991
threefold during oocyte maturation (Luke and Bogenhagen, 1989). At present, we can only offer speculation regarding a rationale for this regulation of the form and type of topoisomerases in oocytes and somatic cells. It may be that a high ratio of topoisomerase II to topoisomerase I is important for the rapid cycles of chromosome condensation and decondensation that accompany early embryogenesis. If this is the case, the additional domains present in the 165-kDa topoisomerase I may simply serve to target its developmentally regulated destruction. It may be that the 165-kDa topoisomerase I contains a domain (or domains) that directs the enzyme to different activities in oocytes compared to those in somatic cells. The amphibian oocyte is a highly differentiated cell. Topoisomerase I is particularly concentrated in the nucleolus (Fleishmann et ah, 1984; Muller et al., 1985). The rapid rates of rRNA synthesis in oocytes may require a specialized topoisomerase I. Ribosomal transcription during oogenesis proceeds at a nearly maximal rate (Davidson, 1986). Topoisomerase I binding sites are closely spaced on ribosomal DNA in X Zaevis oocytes, occurring at intervals of 200 bp (Culotta and SollnerWebb, 1988). Recently topoisomerases have been implicated in the maintenance of genome stability (Wang, 1990). Topoisomerase I is involved in suppression of recombination of the highly repetitive ribosomal gene loci in yeast (Christman, et al. 1989; Kim and Wang, 1989b). When genetic manipulations are performed to limit the topoisomerase activity, copies of rDNA can be excised from the yeast genome as extrachromosomal circles. Amphibian oocytes amplify extrachromosomal copies of rDNA to such an extent that a stage IV oocyte contains -2.5 X lo6 copies of the rDNA repeating unit (Thiebaud, 1979; Davidson, 1986). Little is known about the initial stages in amplification of rDNA. It is possible that a modified form of topoisomerase I is necessary to allow this rDNA amplification. This research was supported by NIH Grant GM 29681to D.F.B. and by NIH Training Grant support for R.E.R. We thank Paul Fisher and Rolf Sternglanz for review of the manuscript prior to publication. We also thank May Luke for helpful suggestions and discussion during the course of these studies. REFERENCES A’ITARDI, D. G., DEPAOLIS,A., and TOCCHINI-VALENTINI,G. P. (1981). Purification and characterization of Xelzopus Zuevis type I topoisomerase. J. Biol. Chem. 256,3654-3661. BRILL, S. J., DINARDO, S., VOELKEL-MEIMAN,K., and STERNGLANZ,R. (1987). Need for DNA topoisomerase activity as a swivel for DNA replication and for transcription of ribosomal RNA. Nature 326, 414-416. BRILL, S. J., and STERNGLANZ,R. (1988). Transcription-dependent
RICHARDAND BOGENHAGEN
X laevis Topoisarnerase I Is Tissue Specific
DNA supercoiling in yeast DNA topoisomerase mutants. Cell 54, 403-411. CHRISTMAN,M. F., DIETRICH, F. S., and FINK, G. R. (1989). Mitotic recombination in the rDNA of S. cerevisiae is suppressed by the combined action of DNA topoisomerase I and II. Cell 55,413-425. CHOW,K., and PEARSON,G. D. (1985). Adenovirus infection elevates levels of cellular topoisomerase I. Proc. Natl. Acud. Sci. USA 82, 2247-2251. CHUNG,T. D., DRAKE, F. H., TARR, K. B., PER, S. R., CROOK,S. T., and MIRABELLI, C. K. (1989). Characterization and immunological identification of cDNA clones encoding two human DNA topoisomerase II genes. Proc. Natl. Acad. Sci. USA 86,9431-9435. CULOTTA,V., and SOLLNER-WEBB,B. (1988). Sites of topoisomerase I action on X. lae7li.sribosomal chromatin: Transcriptionally active rDNA has an -200 bp repeating structure. Cell 52,585-597. D’ARPA, P., MACHLIN, P. S., RATRIE, H., III, ROTHFIELD,N. F., CLEVELAND, D. W., and EARNSHAW,W. C. (1988). cDNA cloning of human DNA topoisomerase I: Catalytic activity of 67.7-kDa carboxyl-terminal fragment. Proc. Natl. Acad. Sci. USA 85,2543-2547. DAVIDSON,E. H. (1986). “Gene Activity in Early Development.” Academic Press, New York. DIGNAM, J. D., LEBOVITZ,R. M., and ROEDER,R. G. (1983). Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11, 1475-1489. FLEISHMANN, G., PFLUGFELDER,G., STEINER, E., JAVAHERIAN, K., HOWARD,G., WANG, J., and ELGIN, S. (1984). DNA topoisomerase I is associated with transcriptionally active regions of the genome. P/w Nutl. Acud. Sci. USA 81, 69586962. HARLOW, E., and LANE, D. (1988). “Antibodies: A Laboratory Manual.” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. HOLT, G. D., and HART, G. W. (1986). The subcellular distribution of terminal N-acetylglucosamine moieties. J. Biol. Chem. 261, 80498057. JAVAHERIAN, K., TSE, Y.-C., and VEGA, J. (1982). Drosophila topoisomerase I: Isolation, purification, and characterization. Nucleic Acids Res. 10, 6945-6955. JUAN, C.-C., HWANG, J., LIU, A.-A., WHANG-PENG,J., KNUTSEN, T., HUEBNER,K., CROCE,C. M., ZHANG, H., WANG, J. C., and LIU, L. F. (1988). Human DNA topoisomerase I is encoded by a single-copy gene that maps to chromosome region 2Oq12-13.2.Prac. N&l. Acad. Sci. USA 85, 8910-13. KAISERMAN,H. B., INGEBRITSEN,T. S., and BENBOW,R. M. (1988). Regulation of Xenopls la&s DNA topoisomerase I activity by phosphorglation in vitro. BiochemistmJ 27, 3216-3222. KIM, R. A., and WANG, J. C. (1989a). Function of DNA topoisomerases as replication swivels in Saccharomyces cerevisiae. J. Mol. Biol. 208, 257-267. KIM, R. A., and WANG, J. C. (198913).A subthreshold level of DNA topoisomerases leads to the excision of yeast rDNA as extrachromosomal rings. Cell 57, 975-985. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.
11
LEE, C., LEVIN, A., and BRANTON,D. (1987). Copper staining: A fiveminute protein stain for sodium dodecyl sulfate-poiyacrylamide gels. Anal. Rio&em. 166,308-312. LUKE, M., and BOGENHAGEN,D. F. (1989). Quantitation of type II topoisomerase in oocytes and eggs of Xenopus laevis. Dev. Biol. 136, 459-468. MARTIN, S. R., MCCOUBREY,W. K., MCCONAUGHY,B. L., YOUNG,L. S., BEEN, M. D., BREWER,B. J., and CHAMPOUX,J. J. (1983). Multiple forms of rat liver type I topoisomerases. In “Methods in Enzymology” (R. Wu, L. Grossman, and K. Moldave, Eds.), Vol. 100, pp. 137144. Academic Press, SD. MULLER, M., PFUND,W., MEHTA, V., and TRASK,D. (1985). Eukaryotic type I topoisomerase is enriched in the nucleolus and catalytically active on ribosomal DNA. EMBO J. 4,1237-1243. RICHARD,R. E., and BOGENHAGEN,D. (1989). A high molecular weight topoisomerase I from Xenopus laevis ovaries. J. Biol. Chem. 264, 4704-4709. SMITH, D. E., and FISHER, P. A. (1984). Identification, developmental regulation, and response to heat shock of two antigenically related forms of a major nuclear envelope protein in Drosophila embryos: Application of an improved method for affinity purification of antibodies using polypeptides immobilized on nitrocellulose blots. J. Cell Biol. 99,20-28. STEWART,A. F., HERRERA, R. E., and NORDHEIM,A. (1990). Rapid induction of c-fos transcription reveals quantitative linkage of RNA polymerase II and DNA topoisomerase I enzyme activities. Cell 60, 141-149. THIEBAUD, C. H. (1979). Quantitative determination of amplified rDNA and its distribution during oogenesis in Xenopus laevis. Chw mosomu 73, 37. THRASH, C., BANKIER, A. T., BARRELL, B. G., and STERNGLANZ,R. (1985). Cloning, characterization, and sequence of the yeast DNA topoisomerase I gene. Proc. Natl. Acad. Sci. USA 82,4374-4378. TRICOLI,J. V., and KOWALSKI,D. (1983). Topoisomerase I from chicken erythrocytes: Purification, characterization, and detection by a deoxyribonucleic acid binding assay. Biochemistry 22,2025-2031. UEMURA,T., MORINO,K., UZAWA, S., SHIOZAKI,K., and YANAGIDA, M. (1987). Cloning and sequencing of Schizosaccharamyces pan&e DNA topoisomerase I gene, and effect of gene disruption. Nucleic Acids Res. 15,9727-9739. WALLIS, J. W., CHREBET,G., BRODSKY,G., ROLFE,M., and ROTHSTEIN, R. (1989). A hyper-recombination mutation in S. cerevisiae identifies a novel eukaryotic topoisomerase. Cell 58,409-419. WANG, J. C. (1985). DNA topoisomerases. Annu. Rev. Biochem. 54, 665-697. WANG, J. C. (1990). The role of DNA topoisomerases in recombination and genome stability: A double-edged sword? Cell 62, 403-406. WRAY, W., BOULIKAS,T., WRAY, V. P., and HANCOCK,R. (1981). Silver staining of protein in polyacrylamide gels. Anal. Bioch,em. 118,197203. ZHANG, H., WANG, J. C., and LIU, L. F. (1988). Involvement of DNA topoisomerase I in transcription of human ribosomal RNA genes. Proc. Natl. Acud. Sci. USA 85,1060-1064.
BIOLOGY
146,4-11 (1991)
The 165kDa DNA Topoisomerase I from Xenopus Levis Oocytes Is a Tissue-Specific Variant ROBERTE.RICHARDANDDANIEL F. BOGENHAGEN Department of Pharmacological Sciences, State University of New York, Stony Brook, New York 11794 Accepted March 6, I!%1 Two forms of topoisomerase I can be purified from Xenopus 2aevi.s. A protein with a molecular mass of 165 kDa has been identified as topoisomerase I in ovaries (Richard and Bogenhagen, 1989. J. Biol. Chem. 264,4704-4709). When a similar purification is performed using liver tissue, topoisomerase I is purified as a DO-kDa protein. Separate rabbit antisera were raised against oocyte and liver topoisomerase I polypeptides. Each antiserum reacts in immunoblottingor immunoprecipitation procedures only with the tissue-specific topoisomerase I polypeptide against which it was generated. The failure of the antiserum raised against liver topoisomerase I to cross-react with the oocyte enzyme suggests that the smaller topoisomerase I is not derived from the 165-kDa oocyte enzyme by proteolysis. X laevis tissue culture cells lysed and processed in the presence of SDS contain the IlO-kDa form of topoisomerase I. The 165-kDa form of topoisomerase I disappears during oocyte maturation in vitro. @ 1991 Academic PRWS, IX.
We recently purified a topoisomerase I with an unusually large molecular mass of 165 kDa from the ovaries of DNA topoisomerases catalyze changes in DNA topol- X Zaevis (Richard and Bogenhagen, 1989). Prior to this ogy by cleaving and resealing the phosphodiester back- finding, the largest topoisomerase I described had been bone of the DNA molecule (Wang, 1985). In eukaryotes, the 135-kDa enzyme of Drosophila melanogaster (Javathere are two major types of topoisomerases, I and II. herian et al, 1982). We assumed that this difference in The cDNAs of three eukaryotic type I enzymes have molecular weight compared to other purified type I been cloned and sequenced. These include human topoisomerases was due to species-specific variability. (D’Arpa et al, 1988), Saccharomyces cerevisae (Thrash et In order to test this hypothesis, we decided to examine al., 1985), and Schixosaccharomyces pombe (Uemura et other tissues from X Zaevis. Our initial purification of aZ., 1987). The human gene is similar in size and se- topoisomerase I from liver of adult X laevis indicated quence to the two yeast genes. Human topoisomerase I that the 165-kDa ovarian enzyme was not present. appears to be a single copy gene that maps to chromo- Rather, a more conventionally sized type I topoisomersome 20 and encodes a protein of 765 amino acids (Juan ase llO-kDa polypeptide was purified. In this paper we et ab, 1988). show that the llO-kDa form is representative of the preTopoisomerase I is involved in a number of cellular dominant, somatic form of topoisomerase I and is not processes including transcription (Zhang et al., 1988; derived from the 165-kDa topoisomerase I by proteolyBrill and Sternglanz, 1988; Stewart et ah, 1990), replica- sis. The 165-kDa form is restricted to oocytes and disaption (Brill et al., 1987;Kim and Wang, 1989a), and recom- pears during oocyte maturation. bination (Christman et al., 1989; Kim and Wang, 198913). The cell must be able to selectively recruit and modulate MATERIALS AND METHODS topoisomerase activity in each of these processes. Recent observations of topoisomerase heterogeneity sug- Materials gest that modulation of topoisomerase activity may ocPhosphocellulose was purchased from Whatman cur by expression of different forms of topoisomerase (Chung et al., 1989; Wallis et aZ.,1989). Since most stud- (Clifton, NJ); immobilon was from Millipore (Bedford, ies have been performed in yeast and mammalian cell MA); hydroxyapatite (HA) Ultrogel was from IBF; culture systems, little is known about topoisomerase I phenyl-Superose HR lO/lO and Protein A-Sepharose during the development of a higher eukaryote. Xenopus were from Pharmacia LKB Biotechnology (Piscataway, laevis offers the advantages of a well-characterized de- NJ); agarose was from Bethesda Research Laboratories velopmental system, in which biochemical amounts of (Bethesda, MD); BCIP/NBT phosphatase substrate system was from Kirkegaard and Perry Laboratories enzymes can be obtained. INTRODUCTION
001%1606/91 $3.00 Copyright All rights
(c) 1991 by Academic Press. Inc. of reproduction in any form reserved.
4
RICHARD AND BOGENHAGEN
X laevis Topoisomerase
(Gaithersburg, MD); PMSF,l benzamidine-HCl, and prestained protein molecular weight markers were from Sigma (St. Louis, MO). The X Zaevis cell line, A6, was obtained from American Type Culture Collection and propagated in 0.75X NCTC 135 media containing 10% fetal bovine serum. Freund’s complete and incomplete adjuvant were from DIFCO (Detroit, MI). Topoisomerase Assay The assay for topoisomerase I activity and the method to radiolabel topoisomerase I were performed as published (Richard and Bogenhagen, 1989). Purification
of Liver Topoisomerase I
The preparation of a nuclear extract from liver employed a variation of the method of Dignam et al. (1983). All operations were carried out at 4°C. Livers were removed from matureX laevis, washed and minced in PBS with 2 mM DTT, 0.4 mM PMSF, 1 mM benzamidineHCl. The tissue was placed in 4 vol of a hypotonic solution consisting of 10 mM Hepes, pH 7.9, 1.5 mM MgCl,, 10 mM KCl, 1 mM DTT, 0.4 mM PMSF, 1 mM benzamidine-HCl, 2.5 pg/ml aprotinin, 2.5 pg/ml leupeptin for 15 min and homogenized with a motor driven glassTeflon homogenizer. Nuclei were pelleted by spinning at 2000 rpm in a Beckman JA-14 centrifuge. The supernatant was discarded. The nuclear pellet was homogenized and resuspended in 5 vol of 20 mM Hepes, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl,, 0.2 mM EDTA, 0.4 mM PMSF, 1 mM DTT, 1 mM benzamidine-HCl, 2.5 pg/ml aprotinin, 2.5 pg/ml leupeptin and spun in a Beckman Ti 70 rotor at 40k rpm for 1 hr. The supernatant (nuclear extract SlOO) was frozen and stored at -80°C (fraction I). The frozen extract was diluted with Buffer A (20 mM Tris-HCl, pH 7.5, 0.2 mM EDTA, 15% glycerol, 1 mM DTT, 1 mM benzamidine-HCl, 0.4 mM PMSF, 1 pg/ml leupeptin, 1 pg/ml aprotinin) to a NaCl concentration of 0.2 M. The protease inhibitors at the indicated concentrations were included in all subsequent buffers. The diluted extract was loaded on a phosphocellulose column (2.5 X 15-cm) and washed with 0.5 MKCl in Buffer A. A 1.2 M KC1 step with Buffer A lacking EDTA was performed to elute topoisomerase I activity (fraction II). This high salt step was loaded directly on a 5 ml HA Ultrogel column (1.6 x 4-cm) equilibrated in Buffer A without EDTA. Topoisomerase I activity was step ’ Abbreviations used: SDS, sodium dodecyl sulfate; PMSF, phenylmethanesulfonyl fluoride; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; Hepes, N-hydroxyethylpiperizine-W-2ethanesulfonic acid; EDTA, ethylenediaminetetraacetic acid; PBS, phosphate-buffered saline; TCA, trichloroacetic acid.
I Is Tissue Specific
5
eluted with 0.8 M KP,, pH 6.9 (fraction III). The step from the hydroxyapatite column was adjusted with 3 M (NH&SO, to a final conductivity equivalent to that of 1.3 M (NH&SO,. This was filtered and loaded on the fast protein liquid chromatography column phenyl-Superose HR lO/lO and eluted with a gradient of (NH&SO, decreasing from 1.3 to 0.1 M. Topoisomerase I activity eluted at approximately 0.8 M (NH&SO, (fraction IV). Generation of Polyclonal Antisera Methods were similar to those described in Harlow and Lane (1988). New Zealand White rabbits were used to generate polyclonal antisera against the 165-kDa ovarian topo I and the llO-kDa liver topo I. Fraction IV from the liver purification was used to gel purify the llO-kDa form. The SDS-gel filtration fraction described by Richard and Bogenhagen (1989) was used to gel purify the 165-kDa form. Proteins were run on SDSPAGE (Laemmli, 1970), identified by copper staining (Lee et al., 1987), excised from the gel, and homogenized in PBS/50% complete Freund’s adjuvant (for the initial injection, subsequent injections were performed with incomplete Freund’s adjuvant). The popliteal lymph nodes were the sites of all injections. Between 5 and 15 pg of protein was used for each injection. Anti-llO-kDa antisera was routinely used at a dilution of l/2000. Anti-165-kDa antisera was used at a dilution of l/10000. When affinity-purified antiserum was required, the method of Smith and Fisher (1984) was employed with minor changes. Immobilon membranes were substituted for nitrocellulose and the NH,SCN washes were omitted. Antibody directed against the 165-kDa polypeptide was affinity-purified from a preparative blot of ovarian fraction III protein. Antibody against the llOkDa form was affinity-purified from a preparative immunoblot of A6 tissue culture cell extract chromatographed on an SDS-hydroxyapatite column (see below). Immunoprecipitation
of Topoisomerase I Activity
Protein A-Sepharose was loaded with IgG by incubating the resin on a rotator with an equal volume of antiserum for 2 hr at 4”C, removing the antiserum, and repeating the procedure. The beads were washed with IP Buffer (20 mM Tris, pH 7.6,150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 0.4 mM PMSF, 1 mM benzamidineHCl) and stored in IP Buffer with 0.02% NaN, at 4°C. Immunoprecipitations were carried out in IP Buffer on a rotator, the beads spun out, and the supernatant was assayed for topoisomerase I activity.
6 Denaturing
DEVELOPMENTAL BIOLOGY
TABLE 1 PURIFICATION OF TYPE I TOPOISOMERASE FROM X laevis LIVER
Lysis of X laevis A6 Tissue Culture Cells
Cells grown on monolayer were removed from T-150 tissue culture flasks (~10’ cells) with versene, pelleted, resuspended in lysis buffer (2% SDS, 50 mM Tris-HCl, pH 7.6, 10 mM DTT), and boiled for 10 min. DNA was sheared by passing the sample through a 26-gauge needle. The extract was spun at 14,000gfor 10 min, and the supernatant removed, frozen in liquid N,, and stored at -80°C. When used, the extract was thawed, diluted in dilution buffer (50 mM Tris-HCl, pH 7.5,100 mM NaCl, 0.2% SDS, 1 mM benzamidine-HCl, 0.4 mMPMSF), and loaded on a l-ml HA Ultrogel column equilibrated in dilution buffer. The column was washed in 0.1 M NaPi, pH 6.9, and stepped with 0.5 M NaPi, pH 6.9. Fractions from this column were TCA precipitated, separated on 6% SDS-PAGE, transferred to immobilon membranes, and probed with either affinity-purified antibody. Transfer of proteins to immunoblots was performed using passive or electroblot transfer conditions as described in Harlow and Lane (1988).
VOLUME 146,199l
Volume (ml)
Fraction I. II. III. IV.
NE-S100 Phosphocellulose Hydroxyapatite Phenyl-Superose
130
50 9 6
Protein (w) 1070 17.5 9.9 0.1
Activity (units)
Sp Act (U/mg)
Yield (7%)
800,000 500,000 360,000 200,000
750 28500 36000 2 x lo6
100 62.5 45
25
trotransfer, the immobilon membrane was probed with affinity-purified anti-165-kDa antibody. RESULTS
Purification
of Topoisomerase I from Liver
The procedure used to purify topoisomerase I from the liver nuclear extract was essentially the same as that previously used to purify topoisomerase I from ovaries (Table 1). The first chromatographic step, involving stepwise elution from phosphocellulose, provided a 35fold purification with good recovery of activity, but left Immunoprecipitations of Topoisomerase I from the enzyme in a relatively large volume of high salt Detergent-Treated Oocytes buffer. The subsequent hydroxyapatite column was used Stage VI oocytes were collected after treatment with largely to concentrate the enzyme. Step elution was subcollagenase as described in Luke and Bogenhagen stituted for gradient elution from the hydroxyapatite (1989). Maturation was induced by addition of proges- column since gradient elution gave little additional puriterone to a final concentration of 5 pg/ml. At each time fication. The enzyme eluted from hydroxyapatite was point, 100 oocytes were removed to a 1.7-ml microfuge applied to a phenyl-Superose column and eluted with tube and 0.5 vol of extraction buffer (80 mM P-glycerol- solutions of decreasing (NH&SO, concentration. The phosphate, 20 mMEGTA, 15 mMMgCl,, 0.1 MNaCl, 0.2 specific activity of fraction IV is in the range of purified mM PMSF, 2 mM DTT, and 1 pg/ml leupeptin) was type I topoisomerases from other sources (Tricoli and added. The oocytes were then spun at the maximal set- Kowalski, 1983; Martin et al., 1983). This purification ting of a microcentrifuge, 16,000 RCF, for 15 min at 4°C. procedure was designed so that the three columns could The clear supernatant (S16) was removed, mixed with be run quickly, in approximately 7 hr, with minimal hanan equal amount of a solution containing 2% SDS, 10 dling of the eluted fractions. Time consuming topoisomerase I assays were carried out only once, after the mMDTT, 0.2 mMPMSF, 2.5 mMEDTA, 2 mMbenzamidine-HCl, 1 pg/ml leupeptin, and boiled for 5 min. The third chromatographic step. The fraction IV enzyme 165-kDa topoisomerase I present in the SDS denatured was stored frozen as a (NH,),SO, precipitate while the S16 was immunoprecipitated following addition of 3 vol enzyme assay data were collected. A wide range of proof a Triton Buffer (4% Triton X-100, 150 mM NaCl, 50 tease inhibitors were included in all of the buffers. The mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 1 mM benzami- polypeptide pattern on a polyacrylamide gel and topoisomerase I enzyme assays of the phenyl-Superose fracdine-HCl, 0.4 mM PMSF, 1 pg/ml leupeptin) essentially as described in Luke and Bogenhagen (1989). Twenty tions are shown in Fig. 1. The topoisomerase activity microliters of Protein A-Sepharose beads loaded with eluted with the first proteins to elute from the column. the anti-165-kDa enzyme was used for each of the im- The majority of the proteins that adsorbed to the colmunoprecipitations. After washing the beads, 25 ~1 of a umn eluted after fraction 12 and are not shown in Fig. 1. solution loading buffer (4% SDS, 20 mMDTT, 10% glyc- One major polypeptide with a molecular weight of erol, 120 mM Tris-HCl, pH 8.8, with bromophenol blue 110,000 and a second polypeptide with a molecular as a mobility marker) was added. The mixture was weight of 80,000 copurified with the activity (Fig. lA, boiled for 2 min and treated with 0.1 M iodoacetamide. arrowhead). Experiments presented below suggest that The beads were sedimented and the supernatant was this 80-kDa polypeptide is a breakdown product derived loaded onto a SDS-polyacrylamide gel. Following elec- from the IlO-kDa polypeptide. This enzyme is clearly a
RICHARD
A
AND BOGENHAGEN
L 1 2 3 4 5 6 7 6 9 10 1112
160D 116 P 64 D
(Fig. 2). This control demonstrated that the ovary enzyme has the high molecular weight reported previously. Never in the course of our experiments has a 165-kDa polypeptide radiolabeled or coeluted with topoisomerase activity in a fraction of liver enzyme.
46 r=-
Antisera Against the Ovarian Topoisomerases
FIG. 1. Phenyl-Superose HR 1000 chromatography of topoisomerase I from liver. (NH&SO, was added to the enzyme fraction eluted from hydroxyapatite (fraction III) until a final conductivity equivalent to that of 1.3 M (NH&SO, was obtained. The solution was then chromatographed on a phenyl-Superose HR lO/lO column as described under Materials and Methods (A) One hundred-microliter samples of the l-ml fractions eluting from the column were TCA precipitated and treated as described by Laemmli (1970). The samples were then subjected to electrophoresis on 6% SDS-polyacrylamide gels. Silver staining was performed as described by Wray et ul. (1981) after Coomassie staining and destaining. Lane L, 100 ~1 of fraction III. (B) Type I topoisomerase assays of the fractions eluting from the phenyl-Superose HR 10110 column. One microliter of l/20 diluted samples from the marked fractions was assayed in a standard reaction. Lane +, 1 ~1 of similarly diluted load (fraction III) was assayed.
type I topoisomerase. Its activity is independent of ATP, it is unable to decatenate kinetoplast DNA, and it has the same sensitivity to camptothecin as the oocyte topoisomerase I described previously (Richard and Bogenhagen, 1989; data not shown). We employed the method of radiolabeling topoisomerase I as outlined previously to identify the polypeptides capable of covalently binding to DNA (Richard and Bogenhagen, 1989). This method specifically labels topoisomerases (Chow and Pearson, 1985). Liver topoisomerase I purified only through fraction III was used in this experiment in order to provide a sufficiently concentrated preparation at the earliest possible purification step, thereby minimizing the possibility of degradation during additional purification. Proteins were denatured by addition of SDS, renatured, incubated with radiolabeled DNA, and then digested with DNAase I (Fig. 2). Only two proteins were labeled by this procedure despite the complex set of polypeptides present in fraction III (see Fig. lA, lane “L,” for polypeptide composition). These DNA-labeled polypeptides have the same molecular weights as the two polypeptides that coeluted with topoisomerase I activity in Fig. 1. Radiolabeling of liver fraction IV proteins resulted in the same pattern (data not shown). As a control, fraction III from an ovarian preparation (Richard and Bogenhagen, 1989) was treated similarly and run on the same gel
and Liver Type I
The purifications mentioned above and in our previous report produced sufficient quantities of gelpurified liver and ovarian topoisomerase I to generate specific polyclonal antisera in rabbits. These two antisera were then used to study the possibility of shared antigenic determinants between the two polypeptides and their respective breakdown products. On protein blots, each antiserum recognized the form of topoisomerase I against which it was raised (Fig. 3), but failed to cross-react with topoisomerase I from the other source. Although neither antiserum neutralizes enzyme activity, each antiserum was capable of immunoprecipitating enzyme activity from the appropriate tissue, as shown in Fig. 4. In agreement with the immunoblotting results, the antisera were not able to immunoprecipitate activity from extracts derived from the other source tissue, i.e., anti-ovarian topoisomerase I antibody did not recognize liver activity and vice versa. These results indicate
Liver
Ovary
180D
116D 84
b
48
L>
of DNA topoisomerase I. The radioFIG. 2. Active site radiolabeling labeling method described under Materials and Methods was used to identify the molecular weight of the type I topoisomerase in fraction III. A. A 50-4 sample of fraction III, processed to radiolabel topoisomerase I, was TCA precipitated, fractionated on 6Y0 SDS-polyacrylamide gel, and subjected to autoradiography. (B) An 80-J sample of fraction III from an ovarian preparation (Richard and Bogenhagen, 1989) was treated as in A. In both lanes the dark band at the bottom represents degraded DNA at the dye front.
8
DEVELOPMENTAL BIOLOGY
VOLUME 146. 1991
ovarian extracts (data not shown). In Fig. 5B, a duplicate immunoblot probed with the affinity-purified anti165kDa antibodies revealed no specific staining. This analysis confirms that tissue culture cells contain the llO-kDa form of topoisomerase I. We estimate that if the 165-kDa polypeptide were present in liver and/or tissue culture cells at a level > 5% of the llO-kDa form it would have been identified by the methods we employed. The 165kDa Form of Topoisomerase I Disappears upon Oocyte Maturation
FIG. 3. Immunoblotting of the 165. and IlO-kDa forms of topoisomerase I with antisera raised against each form. Liver extract (75 ~1 of fraction III) and ovarian extract (200 11 of fraction III) were TCA precipitated, divided equally into two lanes on a 6% SDS-polyacrylamide gel, subjected to electrophoresis, and electroblotted to an immobilon membrane. Lanes marked 1 contain liver extract, lanes marked 2 contain ovarian extract. (A) The immunoblot was probed with antisera raised against the 165.kDa type I topoisomerase. (B) The immunoblot was probed with the antisera raised against the DOkDa form.
that these polyclonal antisera recognize distinct epitopes not shared by the two forms of topoisomerase I.
Since we were not able to detect the 165-kDa topoisomerase I in somatic cells, it appears to be an oocytespecific form of the enzyme that is lost during early development. The Xenopus system provides an opportunity to follow the fate of stored oocyte proteins. We prepared extracts from stage VI oocytes after they had been incubated either in the absence or in the presence of progesterone to induce oocyte maturation. Oocyte maturation results in germinal vesicle breakdown and development of the oocyte into an unfertilized egg. These extracts were processed to immunoprecipitate the 165-kDa form of topoisomerase I, and the protein was detected by immunoblotting. As shown in Fig. 6, the disappearance of the 165-kDa topoisomerase I coincides with oocyte maturation. We have not been able to detect A
anti-110 Enzyme Preim. Immune
-
-
Liver 20 -
40 -
10
20
* 40
ovary 20 -
40 10 -
20
40
-
-
Liver 20 -
40 -
10
20
40
o-=-Y 20 -
40 -
10
20
40
X laevis Tissue Culture Cells Contain the IlO-kDa Topoisomerase I
The possibility existed that the 165kDa form was present in liver but could not be isolated intact. We believed this not to be the case since we observed no crossreactivity between the antibodies directed against the liver and oocyte topoisomerases. Nevertheless, we decided to analyze topoisomerase I present in X laevis tissue culture cells as another somatic cell type. Proteolysis in these cells should be significantly less than in liver preparations. To limit proteolysis, the cells were lysed in the presence of 2% SDS, and the enzyme was concentrated by chromatography on hydroxyapatite in the presence of SDS and subjected to SDS-PAGE. Immunoblots were probed with both antisera following affinity purification. This experiment showed that only the llO-kDa form of topoisomerase I is present in X laevis tissue culture cells (Fig. 5A). The absence of the 80-kDa form suggests that this method of purification successfully limited degradation. The elution conditions employed effectively concentrate the 165-kDa form from
B
anti-185 Enzyme Preim. I-une
FIG. 4. Immunoprecipitation of topoisomerase I activity from liver and ovary fractions. Twenty units of topoisomerase I from fraction III of a liver preparation or from fraction III of an ovarian preparation was incubated with IgG fractions bound to Protein A-Sepharose beads. Ten, twenty, or forty microliters of beads was added to the fractions as marked. One-twentieth of the immunoprecipitation supernatant was added to the topoisomerase I reactions shown in this figure. (A) Immunoprecipitations performed with the anti-llO-kDa antisera with either liver or ovarian activity added as marked. (B) Immunoprecipitations performed with anti-165kDa antisera. (Preim. stands for added preimmune serum).
X lneris Topoisowuvuse I Is Tissue Specilc
RICHARDANDBOGENHAGEN
A
B LFt
w
0.5 $?
L
Ft
W
e 0.5
$
160 11684r 58 489
FIG. 5. X. Irter.is tissue culture cells contain the IlO-kDa somatic form of topoisomerase I. X lof~c+s tissue culture cells were processed as under Materials and Methods, and fractions from the SDS-hydroxyapatite column were separated on a 6%1 SDS-polyacrylamide gel. The samples were transferred to immohilon membranes and probed with either the anti-llO-kDa (A) or anti-165-kDa (B) antisera following affinity purification as described under Materials and Methods. Lane L contains the column load; lane Ft contains the flow through; and lane W contains the column wash. The lane marked “Liver” contains 50 ~1 of fraction III of a liver preparation; the lane marked “Ovary” contains 25 ~1 of fraction III of an ovarian prcparation. Approximately 1/20th of the total 0.5 MNaP, step was loaded in lane 0.3 (lo7 cells worth of topoisomerase I). Similar amounts of protein were added in each lane as determined by staining with Coomassir blue (-1/200th of total load and flow through are loaded lane L and lane Ft). The 80-kDa breakdown product found in the liver prcparation is marked with an asterisk (*).
the llO-kDa form of topoisomerase I in unfertilized eggs either using immunoprecipitation as in Fig. 6 or using a chromatographic technique similar to that used for cultured cells in Fig. 5 (data not shown). These results indicate that the 165-kDa oocyte-specific topoisomerase I is not a storage form of the enzyme for use in embryonic development.
9
than 165 kDa and possess topoisomerase I activity (Richard and Bogenhagen, 1989). These polypeptides are recognized by the anti-165kDa antiserum and do not comigrate with the somatic enzyme on SDS-PAGE (unpublished observations). We believe that the smaller polypeptides described in our previous paper are degradation products of the 165-kDa oocyte form. We have considered the possibility that the discrepancy in molecular weight between ovarian and liver topoisomerase I was due to extensive proteolysis of the 165-kDa form in liver homogenates. Topoisomerase I is subject to proteolysis when purified from liver (Martin et al., 1983). Nevertheless, we consider that the molecu-
116 *
B
I
40T
1
20 -c
/ /
0
DISCIJSSION
Recently, we published the purification of a high molecular weight topoisomerase I from X Zaevis ovaries (Richard and Bogenhagen, 1989). In this paper, we used a similar purification protocol to identify a type I topoisomerase with a molecular weight of 110 kDa from liver. To our knowledge, this work represents the first report of a tissue-specific variation of topoisomerase I. Chung et nl. (1989) recently described a second variant of type II topoisomerase in human cells. Whether these enzymes are differentially expressed in different tissues has not been established. Attardi ef (xl. (1981) and Kaiserman ef al. (1988) have purified forms of topoisomerase I with molecular weights of approximately 67 and 110 kDa, respectively, from X 1ac~i.s ovaries. We have also described polypeptides derived from ovarian extracts that are smaller
i
e-0 0
3
Hours
’
g
FIG. 6. The 165-kDa form of topoisomerase I is lost during oocyte maturation. (A) Immunoblot of topoisomerase I immunoprecipitated with the anti-l65-kDa antiserum. One thousand stage VI oocytes were collected after collagenase treatment of ovarian tissue as described (Luke and Bogenhagen, 1989). Half of the sample was treated with progesterone (lanes labeled +) while the remaining half served as the untreated control (lanes labeled -). One hundred oocytes were removed after 0,3,6 or 9 hr of incubation, and were processed to an S16 extract as described under Materials and Methods and by Luke and Bogenhayen (submitted). Topoisomerase I was immunoprecipitated with the anti-165.kDa antiserum and detected by SDS-PAGE and immunoblotting as described under Materials and Methods. Lane M contains mobility markers of prestained cu,-macroglobulin (180 kDa) and @-galactosidase (116 kDa). Lane C contains a positive control of 500 units of ovarian topoisomerase I purified through step III as described in Richard and Bogenhagen (1989). Lanes labeled “0” contain samples immunoprccipitated with preimmune or anti-165.kDa antiserum from freshly isolated oocytes. (B) Oocytes were scored for the percentage exhibiting white spots as a function of the time of i?r c,itro incubation. White spot formation is a marker of germinal vesicle breakdown and oocyte maturation.
10
DEVELOPMENTALBIOLOGY
lar weight difference between the oocyte and the somatic type I topoisomerases reflects a real difference in the form of the enzyme for three reasons. First, particular care was taken to complete the purification in the shortest possible time in the presence of a wide variety of protease inhibitors. Labeling of the liver enzyme with DNA showed no evidence of 165-kDa topoisomerase I from this source (Fig. 2). Second, the lack of cross-reactivity between the two antisera suggested that the llOkDa form is not a degradation product, but rather an alternate form of the enzyme (Figs. 3 and 4). The fact that the anti-165-kDa antiserum does not recognize the liver form of topoisomerase I might not be noteworthy if the major epitopes of the protein recognized by the antiserum were in a domain removed by proteolysis. However, this sort of argument cannot explain the inability of the anti-llO-kDa antiserum to recognize the oocyte form of topoisomerase I. Finally, we also analyzed the size of topoisomerase I from another somatic cell type not known for proteolysis, X Zaevis tissue culture cells. Extracts produced from tissue culture cells lysed in the presence of SDS contained only the llO-kDa form of the enzyme (Fig. 5). A similar immunoblot probed with the anti-165-kDa antisera showed no specific bands. What is the Relationship between Oocyte and Somatic Forms of Topoisomerase I? The large difference in molecular weight between the oocyte and the somatic forms of topoisomerase I is unlikely to result from differential post-translational modification. The 165-kDa form of topoisomerase I is a phosphoprotein, but complete removal of phosphates by potato acid phosphatase produces only a slight increase in gel mobility (R.E.R., unpublished observation). The 165-kDa form of the enzyme does not appear to be modified by 0-glycosylation (R.E.R., unpublished observation; Holt and Hart, 1986). It seems most likely that these two forms of the enzyme are derived from different mRNAs. There may be two differentially regulated genes for topoisomerase I in the X. laevis genome. Alternatively, the two forms of enzyme might result from tissue-specific differences in the splicing of a primary transcript. Experiments to characterize cDNAs for these two forms of topoisomerase I are in progress. Possible Roles for the Oocyte-SpeciJic X5-kDa Topoisomerase I The oocyte-specific nature of the 165kDa form of topoisomerase I is further emphasized by the observation that this form of the enzyme is specifically degraded during oocyte maturation (Fig. 6). This situation presents an interesting contrast to the observation that the quantity of type II topoisomerase increases nearly
VOLUME146. 1991
threefold during oocyte maturation (Luke and Bogenhagen, 1989). At present, we can only offer speculation regarding a rationale for this regulation of the form and type of topoisomerases in oocytes and somatic cells. It may be that a high ratio of topoisomerase II to topoisomerase I is important for the rapid cycles of chromosome condensation and decondensation that accompany early embryogenesis. If this is the case, the additional domains present in the 165-kDa topoisomerase I may simply serve to target its developmentally regulated destruction. It may be that the 165-kDa topoisomerase I contains a domain (or domains) that directs the enzyme to different activities in oocytes compared to those in somatic cells. The amphibian oocyte is a highly differentiated cell. Topoisomerase I is particularly concentrated in the nucleolus (Fleishmann et ah, 1984; Muller et al., 1985). The rapid rates of rRNA synthesis in oocytes may require a specialized topoisomerase I. Ribosomal transcription during oogenesis proceeds at a nearly maximal rate (Davidson, 1986). Topoisomerase I binding sites are closely spaced on ribosomal DNA in X Zaevis oocytes, occurring at intervals of 200 bp (Culotta and SollnerWebb, 1988). Recently topoisomerases have been implicated in the maintenance of genome stability (Wang, 1990). Topoisomerase I is involved in suppression of recombination of the highly repetitive ribosomal gene loci in yeast (Christman, et al. 1989; Kim and Wang, 1989b). When genetic manipulations are performed to limit the topoisomerase activity, copies of rDNA can be excised from the yeast genome as extrachromosomal circles. Amphibian oocytes amplify extrachromosomal copies of rDNA to such an extent that a stage IV oocyte contains -2.5 X lo6 copies of the rDNA repeating unit (Thiebaud, 1979; Davidson, 1986). Little is known about the initial stages in amplification of rDNA. It is possible that a modified form of topoisomerase I is necessary to allow this rDNA amplification. This research was supported by NIH Grant GM 29681to D.F.B. and by NIH Training Grant support for R.E.R. We thank Paul Fisher and Rolf Sternglanz for review of the manuscript prior to publication. We also thank May Luke for helpful suggestions and discussion during the course of these studies. REFERENCES A’ITARDI, D. G., DEPAOLIS,A., and TOCCHINI-VALENTINI,G. P. (1981). Purification and characterization of Xelzopus Zuevis type I topoisomerase. J. Biol. Chem. 256,3654-3661. BRILL, S. J., DINARDO, S., VOELKEL-MEIMAN,K., and STERNGLANZ,R. (1987). Need for DNA topoisomerase activity as a swivel for DNA replication and for transcription of ribosomal RNA. Nature 326, 414-416. BRILL, S. J., and STERNGLANZ,R. (1988). Transcription-dependent
RICHARDAND BOGENHAGEN
X laevis Topoisarnerase I Is Tissue Specific
DNA supercoiling in yeast DNA topoisomerase mutants. Cell 54, 403-411. CHRISTMAN,M. F., DIETRICH, F. S., and FINK, G. R. (1989). Mitotic recombination in the rDNA of S. cerevisiae is suppressed by the combined action of DNA topoisomerase I and II. Cell 55,413-425. CHOW,K., and PEARSON,G. D. (1985). Adenovirus infection elevates levels of cellular topoisomerase I. Proc. Natl. Acud. Sci. USA 82, 2247-2251. CHUNG,T. D., DRAKE, F. H., TARR, K. B., PER, S. R., CROOK,S. T., and MIRABELLI, C. K. (1989). Characterization and immunological identification of cDNA clones encoding two human DNA topoisomerase II genes. Proc. Natl. Acad. Sci. USA 86,9431-9435. CULOTTA,V., and SOLLNER-WEBB,B. (1988). Sites of topoisomerase I action on X. lae7li.sribosomal chromatin: Transcriptionally active rDNA has an -200 bp repeating structure. Cell 52,585-597. D’ARPA, P., MACHLIN, P. S., RATRIE, H., III, ROTHFIELD,N. F., CLEVELAND, D. W., and EARNSHAW,W. C. (1988). cDNA cloning of human DNA topoisomerase I: Catalytic activity of 67.7-kDa carboxyl-terminal fragment. Proc. Natl. Acad. Sci. USA 85,2543-2547. DAVIDSON,E. H. (1986). “Gene Activity in Early Development.” Academic Press, New York. DIGNAM, J. D., LEBOVITZ,R. M., and ROEDER,R. G. (1983). Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11, 1475-1489. FLEISHMANN, G., PFLUGFELDER,G., STEINER, E., JAVAHERIAN, K., HOWARD,G., WANG, J., and ELGIN, S. (1984). DNA topoisomerase I is associated with transcriptionally active regions of the genome. P/w Nutl. Acud. Sci. USA 81, 69586962. HARLOW, E., and LANE, D. (1988). “Antibodies: A Laboratory Manual.” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. HOLT, G. D., and HART, G. W. (1986). The subcellular distribution of terminal N-acetylglucosamine moieties. J. Biol. Chem. 261, 80498057. JAVAHERIAN, K., TSE, Y.-C., and VEGA, J. (1982). Drosophila topoisomerase I: Isolation, purification, and characterization. Nucleic Acids Res. 10, 6945-6955. JUAN, C.-C., HWANG, J., LIU, A.-A., WHANG-PENG,J., KNUTSEN, T., HUEBNER,K., CROCE,C. M., ZHANG, H., WANG, J. C., and LIU, L. F. (1988). Human DNA topoisomerase I is encoded by a single-copy gene that maps to chromosome region 2Oq12-13.2.Prac. N&l. Acad. Sci. USA 85, 8910-13. KAISERMAN,H. B., INGEBRITSEN,T. S., and BENBOW,R. M. (1988). Regulation of Xenopls la&s DNA topoisomerase I activity by phosphorglation in vitro. BiochemistmJ 27, 3216-3222. KIM, R. A., and WANG, J. C. (1989a). Function of DNA topoisomerases as replication swivels in Saccharomyces cerevisiae. J. Mol. Biol. 208, 257-267. KIM, R. A., and WANG, J. C. (198913).A subthreshold level of DNA topoisomerases leads to the excision of yeast rDNA as extrachromosomal rings. Cell 57, 975-985. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.
11
LEE, C., LEVIN, A., and BRANTON,D. (1987). Copper staining: A fiveminute protein stain for sodium dodecyl sulfate-poiyacrylamide gels. Anal. Rio&em. 166,308-312. LUKE, M., and BOGENHAGEN,D. F. (1989). Quantitation of type II topoisomerase in oocytes and eggs of Xenopus laevis. Dev. Biol. 136, 459-468. MARTIN, S. R., MCCOUBREY,W. K., MCCONAUGHY,B. L., YOUNG,L. S., BEEN, M. D., BREWER,B. J., and CHAMPOUX,J. J. (1983). Multiple forms of rat liver type I topoisomerases. In “Methods in Enzymology” (R. Wu, L. Grossman, and K. Moldave, Eds.), Vol. 100, pp. 137144. Academic Press, SD. MULLER, M., PFUND,W., MEHTA, V., and TRASK,D. (1985). Eukaryotic type I topoisomerase is enriched in the nucleolus and catalytically active on ribosomal DNA. EMBO J. 4,1237-1243. RICHARD,R. E., and BOGENHAGEN,D. (1989). A high molecular weight topoisomerase I from Xenopus laevis ovaries. J. Biol. Chem. 264, 4704-4709. SMITH, D. E., and FISHER, P. A. (1984). Identification, developmental regulation, and response to heat shock of two antigenically related forms of a major nuclear envelope protein in Drosophila embryos: Application of an improved method for affinity purification of antibodies using polypeptides immobilized on nitrocellulose blots. J. Cell Biol. 99,20-28. STEWART,A. F., HERRERA, R. E., and NORDHEIM,A. (1990). Rapid induction of c-fos transcription reveals quantitative linkage of RNA polymerase II and DNA topoisomerase I enzyme activities. Cell 60, 141-149. THIEBAUD, C. H. (1979). Quantitative determination of amplified rDNA and its distribution during oogenesis in Xenopus laevis. Chw mosomu 73, 37. THRASH, C., BANKIER, A. T., BARRELL, B. G., and STERNGLANZ,R. (1985). Cloning, characterization, and sequence of the yeast DNA topoisomerase I gene. Proc. Natl. Acad. Sci. USA 82,4374-4378. TRICOLI,J. V., and KOWALSKI,D. (1983). Topoisomerase I from chicken erythrocytes: Purification, characterization, and detection by a deoxyribonucleic acid binding assay. Biochemistry 22,2025-2031. UEMURA,T., MORINO,K., UZAWA, S., SHIOZAKI,K., and YANAGIDA, M. (1987). Cloning and sequencing of Schizosaccharamyces pan&e DNA topoisomerase I gene, and effect of gene disruption. Nucleic Acids Res. 15,9727-9739. WALLIS, J. W., CHREBET,G., BRODSKY,G., ROLFE,M., and ROTHSTEIN, R. (1989). A hyper-recombination mutation in S. cerevisiae identifies a novel eukaryotic topoisomerase. Cell 58,409-419. WANG, J. C. (1985). DNA topoisomerases. Annu. Rev. Biochem. 54, 665-697. WANG, J. C. (1990). The role of DNA topoisomerases in recombination and genome stability: A double-edged sword? Cell 62, 403-406. WRAY, W., BOULIKAS,T., WRAY, V. P., and HANCOCK,R. (1981). Silver staining of protein in polyacrylamide gels. Anal. Bioch,em. 118,197203. ZHANG, H., WANG, J. C., and LIU, L. F. (1988). Involvement of DNA topoisomerase I in transcription of human ribosomal RNA genes. Proc. Natl. Acud. Sci. USA 85,1060-1064.
