DEVELOPMENTAL
BIOLOGY
64,273-283
(1978)
Activity of a DNA Topoisomerase (Nicking-Closing Enzyme) During Sea Urchin Development and the Cell Cycle D. L. POCCIA,*”
D. LEVINE,*
AND J. C. WANG-+
*Department of Biology, State University of New York, Stony Brook, New York 11794and +Department of Biochemistv and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138 Received December 19, 1977;accepted in revised form January 30,1978 An activity which removes superhelical turns from supercoiled DNA has been detected in sea urchin embryo nuclei. It has properties characteristic of eukaryotic DNA topoisomerases (nicking-closing enzymes) and appears to be tightly bound to the chromatin. Total topoisomerase activity per embryo increases in approximate proportion to cell number from fertilization to the prism stage, which suggests that a large store of active enzyme is not present in the unfertilized egg. No activity could be detected in the sperm nucleus. Nuclear topoisomerase activity per unit DNA varies during the synchronous cell cycles of early cleavage, increasing during S phase and then declining through GZ and M. Possible functions of the enzyme are discussed. INTRODUCTION
A protein capable of removing superhelical turns from negatively supercoiled DNA without leaving single-chain breaks in the DNA backbone was initially reported by Wang (1971) in Escherichia coli. The loss of superhelical turns was shown not to be accompanied by changes in the number of helical turns in the DNA duplex itself and it was postulated that the protein acted by introduction of a transient swivel into the DNA molecule (Wang, 1973). Subsequently, such an activity was detected in and partially purified from a number of eukaryotic sources: mouse embryonic (Champoux and Dulbecco, 1972) and L cells (Vosberg and Vinograd, 1976), human KB (Keller and Wendel, 1974) and HeLa cells (Vosberg et al., 1975), Drosophila eggs (Baase and Wang, 1974), rat liver (Champoux and McConaughy, 1976), and calf thymus (Pulleybank and Morgan, 1975). These activities differed from that of E. coli in salt optimum, M2+ requirement, and ability to relax both positive and negative supercoils. The activity has been shown to be enzy‘Author dressed.
to whom
requests
for reprints
be ad-
matic (Keller, 1975; Vosberg and Vinograd, 1976). The enzyme has been referred to as wprotein (Wang, 19711, swivelase (Wang, 1973), untwisting enzyme (Champoux and Dulbecco, 1972), relaxing activity (Keller and Wendel, 1974), relaxation protein (Vosberg et al., 1975), nicking-closing activity (Vosberg and Vinograd, 1975), and DNA topoisomerase (Wang and Liu, 1978). Several functions have been suggested for the enzyme in uiuo, including roles in replication (Wang, 1971; Rosenberg et al., 1976), transcription (Wang, 1973; Higashinakagawa et al., 19771, recombination (Wang and Liu, 1978), and condensation-decondensation of chromatin (Baase and Wang, 1974). The exact function has remained obscure since no mutants in the protein have been isolated. Sea urchin embryos constitute a potentially useful system for investigating the possible role(s) of the enzyme in uiuo. Embryonic nuclei show varying patterns of DNA and RNA synthesis in early development. DNA synthesis rates during cleavage are among the most rapid known in eukaryotes (Hinegardner et aZ., 1964). From blastula to pluteus this rate declines precipi-
273 0012-1606/78/0642-0273$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
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tously. Embryonic nuclei synthesize RNA at a slightly decreasing rate per cell from early cleavage through pluteus (Davidson, 1976). Cleavage stage embryos possess naturally synchronous cell cycles (Hinegardner et al., 1964), both within individuals and throughout the culture, and thus DNA synthesis and mitotic periods can be investigated relative to Gz of interphase. (No G1 is present, with S beginning in telophase). As an initial attempt to correlate enzyme activities with changes in nuclear activity and chromatin structure, we have measured enzyme levels per nucleus and per embryo during normal development and nuclear activity during two of the early synchronous cell cycles. MATERIALS
AND METHODS
Growth and labeling of embryos. Gametes of Strongylocentrotus purpuratus or Lytechinus pi&us were collected after coelomic injection of 0.5 M KCl. Eggs were filtered through cheesecloth and washed three times in Millipore-filtered artificial sea water (Instant Ocean, Eastlake, Ohio). Eggs were fertilized with freshly diluted semen, washed to remove excess sperm, and cultured in 100 vol of artificial sea water. Radioactive cultures were continuously labeled by adding at 0.5 hr postfertilization [3H]thymidine ([methyZ-3H]thymidine, 6 Ci/mmole, Schwarz/Mann) to approximately 1 &i/ml. Embryos to be used for nuclear isolation before hatching were stripped of fertilization membranes by resuspending them at 0.3 hr postfertilization in sea water taken from a freshly hatched culture of blastulas. Embryos were raised at 15°C. Isolation of nuclei and chromatin and cell lysates. Embryos were pelleted at 500 rpm for 2 min in an IEC PR-6000 centrifuge. Pellets were resuspended in 10 to 25 vol of ice-cold 0.8 M dextrose and layered over 10 ml of 1 M dextrose in a 50-ml conical polycarbonate tube. Embryos were spun through the 1 M dextrose layer at 4000 rpm for 3 to 5 min. All subsequent steps were
VOIAJME 64,197&I
carried out in the cold. Nuclei prepared by the ethanol procedure were obtained by washing the dextrose pellet once in 20 vol of 30% ethanol-0.1% Triton X-100 at 0°C and collecting them at 1500 rpm for 3 min. The pellet was lysed in 10 vol of 10% ethanol-0.1% Triton X-100 by covering the centrifuge tube with paralilm and shaking it gently 5 to 10 times. Nuclei were spun down at 2300 rpm for 10 min and washed once in 15% ethanol-0.05% Triton X-100. This procedure is a modification of that of Mazia et al. (1972). It is critical to keep the ethanol solutions cold and thoroughly wash out seawater ions at the dextrose step. Enzyme specific activities of these nuclei or nuclei prepared by the saline-EDTA procedure agreed within 15%. Crude nuclei prepared by the salineEDTA method were obtained by resuspending the dextrose-washed embryos in 0.075 M NaCl-0.025 M NazEDTA-0.1% Triton X-100-0.01 M Tris-HCl (pH 8), homogenizing by Dounce homogenizer for 10 strokes, and collecting the nuclei by centrifugation at 500 g for 10 min. To make chromatin, the crude nuclei were washed once with 10 vol of 0.075 M NaCl-0.025 M NazEDTA-0.01 M Tris-HCl (pH 8)-0.1% Triton X-100 and collected by centrifugation at 1500 g for 10 min. The nuclei were subjected to three more cycles of washing, each time with 10 vol of 0.01 M Tris-HCl (pH 8) and pelleted at 1500 g, 10,OOOg and 10,OOOg for the successive cycles. Resuspended crude chromatin was centrifuged through 1.7 M sucrose-O.01 M Tris-HCl (pH 8) at 20,000 rpm for 3 hr in a Beckman SW 27 rotor. Nuclear pellets were generally taken up in a glycerol medium containing 20% glycerol-0.002 M Na2EDTA-0.01 M P-mercaptoethanol-0.05 M Tris-HCl (pH 8) and sonicated for 2 set at setting No. 4 (low) on a MSE sonifier. Cell lysates were prepared by sonicating dextrose-washed embryos in the glycerol medium plus 0.1% Triton X-100. Enzyme assays. Standard topoisomerase
POCCIA
ET
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reaction mixtures contained 0.20 M NaCl-0.01 M sodium phosphate (pH 7)0.005 M NazEDTA-50 pg/ml of bovine serum albumin-2 pg/ml of supercoiled phage PM2 DNA and variable quantities of nuclear or cell homogenates containing the activity. Reactions were run in 25-~1 volumes in 1.5-ml conical polypropylene tubes. After incubation for 30 min at 30°C reactions were stopped by adding 0.2% SDS, which also serves to deproteinize the DNA. After 10 min at room temperature, 50 ~1 of 50% glycerol-l pg/rnl of ethidium bromide-100 pg/rnl of bromphenol blue was added and samples were kept on ice until electrophoresis. PM2 DNA was prepared according to Espejo and Canelo (1968) and Espejo et al. (1969). To test for ligase activity, reactions were carried out under topoisomerase reaction conditions but PM2 DNA containing one single-strand scission was substituted for the supercoiled DNA. Products were analyzed for covalent closure by alkali CsCl gradient sedimentation (Baase and Wang, 1974). No ligase activity could be detected.
Gel electrophoresis and assay quantitation. Reaction mixtures were assayed by a modification of the procedure of Keller and Wendel (1974). The 1% agarose resolving gel was 90 X 125 X 1.5 mm underlain with a 2.5% agarose gel to hold it in place. Both gels contained 0.040 M Tris-acetate (pH 8.4)-0.02 M sodium acetate-O.002 M NazEDTA-0.5 pg/ml of ethidium bromide. Samples (50 ~1 each) were electrophoresed at constant voltage (110 V) for 2.5 hr in the cold room. After electrophoresis, gels were transilluminated with short-wave uv light. (UV Products, San Gabriel, California) and photographed using Polaroid 55 P/N film for 1 min at f4.5 through a Kodak Wratten filter (No. 23A). The negative was scanned on a Joyce-Loebl Mark IIIC microdensitometer. The peak height was found empirically to be directly proportional to the amount of DNA in a given sample provided the sample volume was constant. In addition, however, unreacted
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PM2 DNA was routinely electrophoresed at 100, 50, and 25% of initial concentration as calibration standards. Deviations from linearity occurred at less than 25% initial DNA. The standard deviation of six replicate assays was &a%. Metrizamide density gradients. Aliquots of sonicated nuclei were mixed with metrizamide stock solutions at 4°C to a final volume of 5 ml. Samples were centrifuged at 4°C in a Beckman SW 65 rotor for 48 hr. Fractions (0.25 ml) were collected at 4°C and aliquots were counted for 3H and assayed for enzyme activity. Refractive indices of the fractions were determined at 20°C and corrected to density by a formula given by Rickwood and Birnie (1975). Radioactivity determinations. Aliquots of cell suspensions or homogenates were precipitated in 5% perchloric acid, collected on Millipore HAWP filters, washed twice with 5% HClO+ and hydrolyzed in 1 ml of 5% HClO( in scintillation vials for 40 min at 80°C. Ten milliliters of scintillation cocktail (4 g of PPO-0.3 g of POPOP-667 ml of toluene-333 ml of Triton X-100) was added and radioactivity was measured in a Searle Mark II Liquid Scintillation System. DNA determinations. DNA was measured by a modification of the procedure of Beers and Wittliff (1975). An equal volume of 0.2% SDS was added to the sample and the solution was incubated at 60°C for 10 min. An equal volume of 2 M NaCl was added to the solution and SDS was precipitated at 0°C for 10 min. The precipitate was collected at 2500 g for 5 min and extracted with 1 M NaCl. The combined supernatants were diluted with 1 M NaCl to an appropriate volume. Sample aliquots were adjusted to give a final NaCl concentration of 0.4 M. Equivalent aliquots were made to contain 50 pg/ml of DNase I (Worthington, No. 6330)-5 m&f MgCL. Standards were sea urchin sperm DNA. All samples were incubated at 37°C for 60 min. After chilling on ice, 0.1 vol of 40 pg/ml ethidium bromide was added. Fluorescence was determined on an Aminco-Bowman
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spectrofluorometer with an excitation wavelength of 365 nm and emission wavelength of 592 nm. DNA concentrations were determined after subtacting background of appropriate blanks and taking the difference between the readings without and with DNase digestion and comparing this to the linear standard curve. Protein determination. Samples were precipitated with 20% trichloroacetic acid at 4’C for 1 hr. Precipitates were spun down at 16,000 g for 30 min and washed three times with cold 95% ethanol. The final pellets were dissolved in 0.1% SDS-l M NaOH at room temperature. The samples were analyzed by the procedure of Lowry et al. (1951) using bovine serum albumin as the standard.
VOLIJME 64, 1978
tions of assay, the activity appears to be of the topoisomerase type. In the absence of significant endonuclease activity which would constitute a competing reaction, topoisomerase activity can be quantitated as either the amount of supercoiled DNA lost or the amount relaxed DNA produced. We define 1 unit (U) of activity as the amount of enzyme necessary to convert 50% of the supercoils to relaxed forms measured by the fraction of PM2 supercoils remaining after reaction under our assay conditions. As seen in Fig. 2, the
RESULTS
Characterization
of the Enzyme Activity
A true topoisomerase activity will convert covalently closed circular DNA from a supercoiled to a relaxed form. Sonicates of sea urchin embryo nuclei were tested for such an activity by incubation with purified phage PM2 DNA consisting of 90% supercoils and 10% nicked molecules. The reaction products were separated by ethidium bromide-agarose gel electrophoresis into three species corresponding in order of increasing mobility to nicked, supercoiled, and relaxed forms in the absence of ethidium (Fig. 1). As increasing amounts of nuclear material are added to the reaction mixture, more supercoiled form becomes converted to the relaxed form. The nicked form increases routinely by less than 10% of its original amount (i.e., less than 1% nicking of supercoils). In addition to topoisomerase activity, it is possible to generate relaxed forms from supercoiled forms by a combination of endonuclease and ligase actions to free the closed circular DNA molecules of the topological constraint (Wang, 1971). Aliquots of nuclei were tested for ligase activity under topoisomerase reaction conditions. Since no ligase was detected under condi-
a.
b.
C.
0 DIRECTION OF MIGRATION FIG. 1. Gel electrophoretogram of PM2 DNA reacted with varying amounts of sea urchin blastula nuclear sonicates containing topoisomerase activity. Products of reaction of PM2 DNA with approximately (a) 0, (b) 0.75, and (c) 1.5 U of blastula nuclear enzyme separated on a 1% agarose-ethidium bromide gel. Species from left to right correspond to nicked, supercoiled, and relaxed DNA in the absence of ethidium (Keller and Wendel, 1974).
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Topoisomerase
NaCl in the incubation is less than 0.05 M or greater than 0.25 M (Fig. 3a). Maximal activity is observed at slightly less than 0.20 M NaCl with a shoulder near 0.10 M. The temperature optimum of the reaction occurs at 30°C with no activity above 50°C (Fig. 3b). Treatment of nuclear sonicates at 100°C for 2 min or with 0.1% sodium dodecyl sulfate completely abolishes activity. The pH optimum centers around neutrality for potassium phosphate buffers (Fig. 3~).
&o.mL E
0
Chromatin Localization 5
IO
AMOUNT ENZYME ADDED t/J) FIG. 2. ation with to reaction lated as a maining.
Proportion of topoisomerase activity variamount of blastula nuclear sonicate added mixture. The amount of activity is calcufraction of the supercoiled DNA peak re-
of the reaction is directly proportional to the amount of nuclear homogenate added up to at least 80% conversion, and there is no significant self-inhibition of the reaction at up to 20 U of excess homogenate added. In an active fraction, the amount of chromatin DNA added for an assay is approximately equal to the amount of supercoiled PM2 DNA reactant. To test for inhibition of the activity by chromatin DNA, increasing amounts of sperm chromatin DNA, which lacks detectable topoisomerase activity (co.5 U/pg), were added to a fixed amount of blastula chromatin (23 U/pg) and the mixtures were assayed. Table 1 shows that there is little inhibition by the sperm chromatin up to a 20-fold DNA excess over PM2 substrate. The sensitivity of the assay is limited by nucleic acids in the homogenates which migrate into the gel at the regions of interest. In practice, however, this is only of concern for sperm nuclei which exhibit no detectable activity and sets an upper limit of activity for these nuclei. Salt, temperature, and pH optima were determined for the nuclear enzyme. No activity is detected if the concentration of
extent
of the Enzyme
Although the activity is detected in nuclear fractions of sea urchin embryos, its presence could be interpreted as a cosedimenting contaminant of the crude nuclei. That this is unlikely is indicated by two types of experiments. First, when our blastula nuclear fraction was purified to chromatin by standard procedures involving salt washes and sedimentation through a 1.7 M sucrose layer, the protein to DNA ratio of the chromatin was one-half that of the nuclei. The ratio of enzyme activity to DNA was only slightly decreased, however (Table 2). Second, if the sonicated blastula nuclei are mixed with metrizamide solutions at low ionic strength (0.05 M Tris-HCl) and centrifuged to equilibrium, the chromatin bands as a single sharp peak at 1.20 g/cc (Fig. 4a), a value previously reported for sheared chromatin (Rickwood and Birnie, TABLE
1
ABSENCEOF~NHIRITION OFTOPOISOMERASE ACTIVITYBYEXCF,SSNLJCLEAR DNA” Sperm nuclear DNA per blastula nuclear DNA 20 6.0 2.0 0.60 0.20 0.00
Percentage supercoiled PM2 DNA remaining 62 70 75 63 70 64
” Varying amounts of sperm nuclei were added to a constant amount of blastula nuclear sonicate and PM2 DNA and the reaction was allowed to proceed. The ratio of blastula nuclear DNA to PM2 DNA in the reaction mixture was 0.80.
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6
7
8
PH
FIG. 3. Enzyme activity optima for blastula nuclear topoisomerase. Reactions were carried out under standard conditions except that (a) NaCl concentration, (b) temperature of incubation, or (c) pH of phosphate buffer was varied. TABLE SPECIFIC
ACTIVITY
Source
Crude nuclei Chromatin
2
OF NUCLEAR ENZYME”
AND CHROMATIN
Units per micro- Micrograms of gram of DNA protein per microgram of DNA 44 38
4.4 2.2
” Crude nuclei made using the saline-EDTA procedure and chromatin were prepared as described under Materials and Methods.
1975). The bulk of the activity (80%) cobands with the bulk of the DNA (90%) in five fractions. No peak of activity is seen at higher densities characteristic of free protein or ribonucleoprotein particles. If the gradient is made 1.0 M in NaCl, however, the activity can be dissociated from the partially deproteinized chromatin (Fig. 4b) and is found at densities characteristic of free protein in metrizamide (Rickwood and Birnie, 1975). These experiments support the proposition that the nuclear enzyme activity is largely bound to chromatin and not a cosedimenting contaminant. We were not able to detect cytoplasmic activity in blastulas after sedimenting nuclei at 10,000 g.
Enzyme Activity During Embryonic Development Enzyme activity per unit DNA was measured for eight-cell, blastula, gastrula, and prism stage nuclei. Enzyme activity per unit protein was measured in whole embryo son-
icates at early cleavage stages, blastula, gastrula, and prism. Since development of the sea urchin up to the prism stage is characterized by a large increase in cell number (and therefore nuclear DNA) but no change in mass (or protein) per embryo, these numbers should be proportional to nuclear activity per cell (or per nucleus) and total activity per embryo respectively. Figure 5 shows a semi-logarithmic plot of cells per embryo, nuclear activity per DNA, or total activity per protein as a function of time of development. Nuclear activity decreases slightly during development, about three-fold from 5 to 72 hr. Total activity increases approximately 400-fold during development, however, roughly parallel to the increase in cell number per embryo. Therefore, it appears that the unfertilized egg contains only a small store of topoisomerase activity (the sperm apparently none) and during embryonic development more activity is produced to keep pace with the increase in the number of cells. The early time points in both graphs show the largest errors but for different reasons. The very low activity per unit protein in whole embryos prior to 10 hr of development is difficult to measure. Interference of endogenous nucleic acid background on the agarose gels limits the amount which can be loaded; at very high inputs of homogenates nuclease action becomes significant. It is clear, however, that a large increase in total activity per embryo
POCCIA
ET
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Sea Urchin
occurs between early cleavage and blastula. Second, variability is found in eight-cell stage nuclei. This is probably due to isolation from different parts of the cell cycle in cultures from different females (see below).
Enzyme Activity during the Cell Cycle To assay the nuclear activity of the enzyme through the cell cycle, a culture was
0
a
5 FRACTION
IO
15
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grown in the presence of [3H]thymidine. Every 15 min, two aliquots were taken. One was immediately precipitated in cold 5% perchloric acid for determination of r3H]thymidine incorporation and the second was washed with cold sea water and immediately used for nuclear isolation. The nuclei were resuspended and aliquots were processed for determination of C3H]thymi-
20
NUMBER
Topoisomerase
0 b
5 FRACTION
IO
I5 NUMBER
4. Equilibrium buoyant density gradients of blastula nuclei in metrizamide. [“H]Tbymidine-labeled FIG. blastula nuclei were adjusted to approximately - 40% (w/v) metrizamide solution containing (a) 0.05 MTris-HCI (pH 8) or (b) 1.0 M NaCl-0.05 M Tris-HCl (pH 8) and centrifuged at 50,000 rpm in a SW 65 rotor at 4°C for 48 hr. Aliquots were counted for acid-precipitable radioactivity and assayed for topoisomerase. HATCHING
GASTRULA
HOURS AFTER FERTILIZATION
FIG. 5. Levels of topoisomerase activity and cells per embryo during sea urchin embryonic development. A plot of cells per embryo for S. purpuratus is included, which was taken from Hinegardner (1967). Time of the first three cleavages, hatching, and gastrulation in the cultures was the same as that given by Hinegardner. Total activity is proportional to activity per embryo. Nuclear activity is proportional to activity per nucleus. Each point represents a separate determination.
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dine content or assay of enzyme activity. The profiles of r3H]thymidine incorporation (Fig. 6) in embryos and in nuclei were of essentially identical shape, being two parallel step functions indicating the degree of synchrony in the culture and showing that the DNA synthesis cycle did not progress significantly during the 13-min period during which the nuclear isolation procedure occurred. The amount of thymidine incorporation in the nuclei was 60 to 80% that of whole embryos, reflecting the yield of nuclei during isolation. The enzyme activity per aliquot, which is proportional to activity per embryo, varies in a characteristic cyclic fashion. Activity increases during the S period and reaches a maximum near the end of S. It decreases during Gz and mitosis and reaches a low point near the beginning of S or at telophase of mitosis since these embryos lack a G1 (Hinegardner et al., 1964). The difference in activity between high and low points may be as much as lo-fold per embryo (or 5-fold per unit DNA since DNA doubles by the end of S), and this number
VOLUME 64, 1976
is probably a minimum since the times of isolation might not be at the exact extremes of the cycle and the embryos are not absolutely synchronous. This experiment was repeated twice, and the phasing of the cyclic enzyme change relative to the thymidine incorporation curve was identical, while the amplitude varied. Mixing experiments with the high and low points of the cycle showed additivity of activity, suggesting that neither an inhibitor nor an activator, diffusible and in excess, is present in either preparation. DISCUSSION
The activity detected in isolated sea urchin nuclei appears to be a typical eukaryotic topoisomerase type on the basis of several criteria: (1) relaxation of supercoiled DNA, (2) absence of significant endonuclease, (3) absence of DNA ligase, (4) a salt optimum of 0.2 M, and (5) absence of M$ requirement. The activity is abolished by 0.1% SDS or heating to 100°C for 5 min. The pH optimum for reaction is similar to that reported for the Drosophila enzyme
TIME POST FERTILIZATION hours) FIG. 6. Levels of nuclear topoisomerase activity during cleavage cell cycles. Nuclei were isolated from samples removed at 15-min intervals from a culture grown in the presence of [“Hlthymidine (1 pCi/ml). Equivalent aliquots of the culture and of the isolated nuclei were acid precipitated to determine thymidine incorporation. Equal aliquots of nuclear sonicates were assayed for enzyme activity so that the amount of nuclear activity in this case is proportional to units per embryo rather than units per nucleus as in Fig. 5. Approximate DNA synthetic periods are indicated at the top of the graph. The arrow indicates time of addition of [‘Hlthymidine.
POCCIA
ET
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Sea Urchin
(Baase and Wang, 1974). The temperature optimum of 30°C and low activity at 37’C may reflect the fact that the sea urchins live and develop at about 15°C. Similar heat sensitivities compared to mammalian enzymes have been reported for sea urchin DNA polymerase (Loeb, 1969) and ribonuclease (Fernlund and Josephsson, 1968). Alternatively, it may reflect a general sensitivity of DNA topoisomerases since the Drosophila activity also shows a temperature optimum near 30°C (Baase and Wang, 1974). The activity present in isolated sea urchin nuclei appears to be fairly tightly bound to the chromatin since it copurifies with the chromatin and bands with chromatin in metrizamide density gradients. Since the nuclei are isolated in the presence of nonionic detergent that strips away most of the nuclear envelope, it is unlikely that the enzyme that we detect is primarily associated with the nuclear membranes (see Rosenberg et al., 1976; Yoshida et al., 1977). The way in which topoisomerase activity is elaborated during sea urchin embryonic development is strikingly different from DNA or RNA polymerase activities. A store of DNA polymerase is present in unfertilized sea urchin egg cytoplasm, and total enzyme activity per embryo remains essentially constant up to gastrula (Fansler and Loeb, 1969; Loeb et al., 1969) while undergoing a gradual shift to the nuclei (Fansler and Loeb, 1972). RNA polymerase II and III activities, which account for the bulk of nuclear RNA synthesis in the early embryo, are also maintained at approximately constant levels per embryo up to gastrulation (Roeder and Rutter, 1970). Other activities associated with DNA synthesis or metabolism remain constant during early development as well, such as DNase (Mazia et al., 1948) and four deoxyribonucleotide kinases (Fansler and Loeb, 1969). In contrast, topoisomerase activity increases per embryo roughly in parallel with the increase in the number of nuclei per
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embryo through the prism stage. We do not know if the increase is due to synthesis or progressive activation of the enzyme. Interestingly, sea urchin RNA polymerase I activity has been reported to increase in proportion to celI number during early development (Roeder and Rutter, 1970). The topoisomerase activity does show at least a lo-fold variation in the nuclei during the cell cycle, increasing during S phase, decreasing during Gz, and reaching a minimum at the end of mitosis. The increase in activity during S probably does not represent a simple cumulative increase in chromatin-associated enzyme due to a duplication of the genes for topoisomerase, since the activity begins to fall immediately after S, although on the average, the proportion of enzyme to nuclear DNA is fairly constant over the early developmental stages up to 72 hr. DNA polymerase shows a cyclic variation in nuclear activity as well but differs from topoisomerase in that it reaches a maximum in the first half of S (Fansler and Loeb, 1972). No correlation is possible with either the overall rate of RNA synthesis or the activity of RNA polymerases through the cell cycle in early sea urchin embryos, since no data have been reported on these subjects. Our results on topoisomerase activity in the cell cycle complement those of Rosenberg et al. (1976), who showed low activities in Go and G1 but a substantial increase in S using extracts of whole cultured cells of WiLZ (human lymphocyte). No Gz or M measurements were made. They suggested that the enzyme might play a role in controlling replication but not transcription because of the low activity in G1. On the other hand, Higashinakagawa et al. (1977) found a topoisomerase enzyme associated with actively transcribing (but not replicating) amplified ribosomal gene chromatin from Xenopus laevis and suggested that it might have a role in transcription. Since a large proportion of protein synthesis in cleaving sea urchin embryos is
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devoted to histones (Kedes, 1976), much of the newly synthesized mRNA accumulation on polysomes is histone mRNA (Nemer et al., 1974), and this accumulation is linked to the S phase of the cell cycle (Kedes and Gross, 1969); the cyclic variation in topoisomerase might be correlated with transcription of histone genes. However, it is not certain that synthesis of histone mRNA sequences represents the bulk of nuclear RNA synthesis in early embryos and recent work with HeLa cells indicates that histone mRNA may be synthesized throughout the cell cycle (Melli et al., 1977). One might expect an enzyme involved in chromosome condensation or decondensation to show a change in activity during mitosis. Our data indicate, however, a steady decline throughout M during which chromosomes are at first condensing and then decondensing. The only nucleus we found totally devoid of activity was the mature sperm nucleus, which is inactive in RNA or DNA synthesis. We are currently attempting to study when and how this nucleus acquires topoisomerase activity in the first cell cycle (which includes a G1 period). We thank Dr. Daniel Mazia for suggesting the ethanol procedure for nuclear isolation and our initial collaboration and Mr. Eric Fairfield for assistance and discussion on the agarose-ethidium gel electrophoretic analyses. This work was supported by grants from the National Institutes of Health HD 09654 to D.L.P. and GM 24544 to J.C.W. REFERENCES BAASF,, W. A., and WANG, J. C. (1974). An w protein from Drosophila melanogaster. Biochemistry 13, 4299-4303. BEEHS, P. C., and WITLIFF, J. L. (1975). Measurement of DNA and RNA in mammary gland homogenates by the ethidium bromide technique. Anal. Biochem. 63,433-441. CHAMPOUX, J. J., and DULFJECCO, R. (1972). An activity from mammalian cells that untwists superhelical DNA-A possible swivel for DNA replication. Proc. Nat. Acad. Sci. USA 69, 143-146. CHAMPOUX, J. J., and MCCONAUGHY, B. J. (1976). Purification and characterization of the DNA untwisting enzyme from rat liver. Biochemistry 15, 4638-4642.
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DAVIDSON, E. H. (1976). “Gene Activity in Early Development,” p. 162, Academic Press, New York. ESPEJO, R. T., and CANELO, E. S. (1968). Properties of bacteriophage PM2: A lipid-containing bacterial virus. Virology 34, 738-747. ESPWO, R. T., CANEI,O, E. S., and SINSHEIMER, R. L. (1969). DNA of bacteriophage PM2: A closed circular double-stranded molecule. Proc. Nat. Acad. Sci. USA 63,1164-116&X FANSLER, B. A., and LOEB, L. A. (1969). Sea urchin DNA polymerase: II, Changing patterns of localization during early development. Exp. Cell Res. 57, 305-310. FANSLER, B. A., and LOEB, L. A. (1972). Sea urchin DNA polymerase: IV, Reversible association of DNA polymerase with nuclei during the cell cycle. Exp. Cell Res. 75,433-441. FERNLUND, P., and JOSEPFSSON, L., (1968). Preparation and properties of sea-urchin ribonuclease. Biochim. Biophys. Acta 151,373-382. HIGASHINAKAGAWA, T., WAHN, H., and RF.EDER, R. H. (1977). Isolation of ribosomal gene chromatin. Develop. Biol. 55, 375-386. HINEGARDNER, R. T. (1967). Echinoderms. In “Methods in Developmental Biology” (F. H. Wilt and N. K. Wessells, eds.), pp. 139-155, Cromwell-Collier, New York. HINEGAHDNEH, R. T., RAO, B., and FELDMAN, D. E. (1964). The DNA synthetic period during early development of the sea urchin egg. Exp. Cell Res. 36, 53-61. KEDES, L. H. (1976). Histone messengers and histone genes. Cell 8,321-331. KEDES, L. H., and GROSS, P. R. (1969). Identification in cleaving embryos of three RNA species serving as templates for the synthesis of nuclear proteins. Nature (London) 223, 1335-1339. KF,LLEH, W. (1975). Characterization of purified DNArelaxing enzyme from human tissue culture cells. Proc. Nat. Acad. Sci. USA 72,2550-2554. KELLER, W., and WF,NDEL, I. (1974). Stepwise relaxation of supercoiled SV40 DNA Cold Spring Harbor Symp. Quant. Biol. 39, 199-208. LOEB, L. A. (1969). Purification and properties of deoxyribonucleic acid polymerase from nuclei of sea urchin embryos. J. Biol. Chem. 244, 1672-1681. LOF,B, L. A., FANSIXH, B., WII,LIAMS, R., and MAZIA, D. (1969). Sea urchin nuclear DNA polymerase: I, Localization in nuclei during rapid DNA synthesis. Exp. Cell Res. 57, 298-304. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MAZIA, D., BLUMENTHAL, G., and BENSON, E. (1948). The activity and distribution of DNase and phosphatases in the early development of Arbaciapunctulata. Biol. Bull. 95,250-251. MAZIA, D., PETZELT, R., WILLIAMS, R. O., and MEZA,
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I. (1972). A Ca-activated ATPase in the mitotic apparatus of the sea urchin egg (isolated by a new method). Exp. Cell Res. 70,325-332. MELLI, M., SPINELLI, G., and ARNOLD, E. (1977). Synthesis of histone messenger RNA of HeLa cells during the cell cycle. Cell 12, 167-174. NEMER, M., GRAHAM, M., and DUBROFF, L. M. (1974). Co-existence of non-histone messenger RNA species lacking and containing polyadenylic acid in sea urchin embryos. J. Mol. Biol. 89,435-454. PULLEYBANK, D. E., and MORGAN, A. R. (1975). The partial purification of “w” protein from calf thymus. Biochemistry 14.5205-5209. RICKWOOD, D., and BIRNIF., G. D. (1975). Metrizamide, a new density gradient medium. FEBS Lett. 50, 102-110. ROF,DER, R. G., and RUTTEH, W. J. (1970). Multiple RNA polymerases and RNA synthesis during sea urchin development. Biochemistry 9,2543-2553. ROSENBERG, B. H., UNGERS, G., and DEIJTSCH, J. F. (1976). Variation in DNA swivel enzyme activity during the mammalian cell cycle. Nucleic Acids Res. 3.3305-3311.
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VOSBERG, H.-P., GROSSMAN, L. I., and VINOGHAD, J. (1975). Isolation and partial characterization of the relaxation protein from nuclei of cultured mouse and human cells. Eur. J. Biochem. 55,79-93. VOSBEHG, H.-P., And VINOGRAD, J. (1976). Puritication and demonstration of the enzymatic character of the nicking-closing protein from mouse L cells. Biochem. Biophys. Res. Commun. 68,456-464. WANG, J. C. (1971). Interaction between DNA and an Escherichia coli protein w. J. Mol. Biol. 55, 523-533. WANG, J. C. (1973). Protein w: A DNA swivelase from E. coZi?Zn “DNA Synthesis in Vitro.” (R. D. Wells and R. B. Inman, eds.), pp. 163-174. University Park Press, Baltimore, Md. WANG, J. C., and LIU, L. F. (1978). DNA topoisomerases: Enzymes which catalyze the concerted breaking and rejoining of DNA backbone bonds. In “Molecular Genetics, Part 3” (H. Taylor, ed.), Acad. Press, New York. in press. YOSHIDA, S., UNC.ERS, G., and ROSENBERG, B. H. (1977). DNA swivel enzyme activity in a nuclear membrane fraction. Nucleic Acids. Res. 4, 223-228.
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(1978)
Activity of a DNA Topoisomerase (Nicking-Closing Enzyme) During Sea Urchin Development and the Cell Cycle D. L. POCCIA,*”
D. LEVINE,*
AND J. C. WANG-+
*Department of Biology, State University of New York, Stony Brook, New York 11794and +Department of Biochemistv and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138 Received December 19, 1977;accepted in revised form January 30,1978 An activity which removes superhelical turns from supercoiled DNA has been detected in sea urchin embryo nuclei. It has properties characteristic of eukaryotic DNA topoisomerases (nicking-closing enzymes) and appears to be tightly bound to the chromatin. Total topoisomerase activity per embryo increases in approximate proportion to cell number from fertilization to the prism stage, which suggests that a large store of active enzyme is not present in the unfertilized egg. No activity could be detected in the sperm nucleus. Nuclear topoisomerase activity per unit DNA varies during the synchronous cell cycles of early cleavage, increasing during S phase and then declining through GZ and M. Possible functions of the enzyme are discussed. INTRODUCTION
A protein capable of removing superhelical turns from negatively supercoiled DNA without leaving single-chain breaks in the DNA backbone was initially reported by Wang (1971) in Escherichia coli. The loss of superhelical turns was shown not to be accompanied by changes in the number of helical turns in the DNA duplex itself and it was postulated that the protein acted by introduction of a transient swivel into the DNA molecule (Wang, 1973). Subsequently, such an activity was detected in and partially purified from a number of eukaryotic sources: mouse embryonic (Champoux and Dulbecco, 1972) and L cells (Vosberg and Vinograd, 1976), human KB (Keller and Wendel, 1974) and HeLa cells (Vosberg et al., 1975), Drosophila eggs (Baase and Wang, 1974), rat liver (Champoux and McConaughy, 1976), and calf thymus (Pulleybank and Morgan, 1975). These activities differed from that of E. coli in salt optimum, M2+ requirement, and ability to relax both positive and negative supercoils. The activity has been shown to be enzy‘Author dressed.
to whom
requests
for reprints
be ad-
matic (Keller, 1975; Vosberg and Vinograd, 1976). The enzyme has been referred to as wprotein (Wang, 19711, swivelase (Wang, 1973), untwisting enzyme (Champoux and Dulbecco, 1972), relaxing activity (Keller and Wendel, 1974), relaxation protein (Vosberg et al., 1975), nicking-closing activity (Vosberg and Vinograd, 1975), and DNA topoisomerase (Wang and Liu, 1978). Several functions have been suggested for the enzyme in uiuo, including roles in replication (Wang, 1971; Rosenberg et al., 1976), transcription (Wang, 1973; Higashinakagawa et al., 19771, recombination (Wang and Liu, 1978), and condensation-decondensation of chromatin (Baase and Wang, 1974). The exact function has remained obscure since no mutants in the protein have been isolated. Sea urchin embryos constitute a potentially useful system for investigating the possible role(s) of the enzyme in uiuo. Embryonic nuclei show varying patterns of DNA and RNA synthesis in early development. DNA synthesis rates during cleavage are among the most rapid known in eukaryotes (Hinegardner et aZ., 1964). From blastula to pluteus this rate declines precipi-
273 0012-1606/78/0642-0273$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
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tously. Embryonic nuclei synthesize RNA at a slightly decreasing rate per cell from early cleavage through pluteus (Davidson, 1976). Cleavage stage embryos possess naturally synchronous cell cycles (Hinegardner et al., 1964), both within individuals and throughout the culture, and thus DNA synthesis and mitotic periods can be investigated relative to Gz of interphase. (No G1 is present, with S beginning in telophase). As an initial attempt to correlate enzyme activities with changes in nuclear activity and chromatin structure, we have measured enzyme levels per nucleus and per embryo during normal development and nuclear activity during two of the early synchronous cell cycles. MATERIALS
AND METHODS
Growth and labeling of embryos. Gametes of Strongylocentrotus purpuratus or Lytechinus pi&us were collected after coelomic injection of 0.5 M KCl. Eggs were filtered through cheesecloth and washed three times in Millipore-filtered artificial sea water (Instant Ocean, Eastlake, Ohio). Eggs were fertilized with freshly diluted semen, washed to remove excess sperm, and cultured in 100 vol of artificial sea water. Radioactive cultures were continuously labeled by adding at 0.5 hr postfertilization [3H]thymidine ([methyZ-3H]thymidine, 6 Ci/mmole, Schwarz/Mann) to approximately 1 &i/ml. Embryos to be used for nuclear isolation before hatching were stripped of fertilization membranes by resuspending them at 0.3 hr postfertilization in sea water taken from a freshly hatched culture of blastulas. Embryos were raised at 15°C. Isolation of nuclei and chromatin and cell lysates. Embryos were pelleted at 500 rpm for 2 min in an IEC PR-6000 centrifuge. Pellets were resuspended in 10 to 25 vol of ice-cold 0.8 M dextrose and layered over 10 ml of 1 M dextrose in a 50-ml conical polycarbonate tube. Embryos were spun through the 1 M dextrose layer at 4000 rpm for 3 to 5 min. All subsequent steps were
VOIAJME 64,197&I
carried out in the cold. Nuclei prepared by the ethanol procedure were obtained by washing the dextrose pellet once in 20 vol of 30% ethanol-0.1% Triton X-100 at 0°C and collecting them at 1500 rpm for 3 min. The pellet was lysed in 10 vol of 10% ethanol-0.1% Triton X-100 by covering the centrifuge tube with paralilm and shaking it gently 5 to 10 times. Nuclei were spun down at 2300 rpm for 10 min and washed once in 15% ethanol-0.05% Triton X-100. This procedure is a modification of that of Mazia et al. (1972). It is critical to keep the ethanol solutions cold and thoroughly wash out seawater ions at the dextrose step. Enzyme specific activities of these nuclei or nuclei prepared by the saline-EDTA procedure agreed within 15%. Crude nuclei prepared by the salineEDTA method were obtained by resuspending the dextrose-washed embryos in 0.075 M NaCl-0.025 M NazEDTA-0.1% Triton X-100-0.01 M Tris-HCl (pH 8), homogenizing by Dounce homogenizer for 10 strokes, and collecting the nuclei by centrifugation at 500 g for 10 min. To make chromatin, the crude nuclei were washed once with 10 vol of 0.075 M NaCl-0.025 M NazEDTA-0.01 M Tris-HCl (pH 8)-0.1% Triton X-100 and collected by centrifugation at 1500 g for 10 min. The nuclei were subjected to three more cycles of washing, each time with 10 vol of 0.01 M Tris-HCl (pH 8) and pelleted at 1500 g, 10,OOOg and 10,OOOg for the successive cycles. Resuspended crude chromatin was centrifuged through 1.7 M sucrose-O.01 M Tris-HCl (pH 8) at 20,000 rpm for 3 hr in a Beckman SW 27 rotor. Nuclear pellets were generally taken up in a glycerol medium containing 20% glycerol-0.002 M Na2EDTA-0.01 M P-mercaptoethanol-0.05 M Tris-HCl (pH 8) and sonicated for 2 set at setting No. 4 (low) on a MSE sonifier. Cell lysates were prepared by sonicating dextrose-washed embryos in the glycerol medium plus 0.1% Triton X-100. Enzyme assays. Standard topoisomerase
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reaction mixtures contained 0.20 M NaCl-0.01 M sodium phosphate (pH 7)0.005 M NazEDTA-50 pg/ml of bovine serum albumin-2 pg/ml of supercoiled phage PM2 DNA and variable quantities of nuclear or cell homogenates containing the activity. Reactions were run in 25-~1 volumes in 1.5-ml conical polypropylene tubes. After incubation for 30 min at 30°C reactions were stopped by adding 0.2% SDS, which also serves to deproteinize the DNA. After 10 min at room temperature, 50 ~1 of 50% glycerol-l pg/rnl of ethidium bromide-100 pg/rnl of bromphenol blue was added and samples were kept on ice until electrophoresis. PM2 DNA was prepared according to Espejo and Canelo (1968) and Espejo et al. (1969). To test for ligase activity, reactions were carried out under topoisomerase reaction conditions but PM2 DNA containing one single-strand scission was substituted for the supercoiled DNA. Products were analyzed for covalent closure by alkali CsCl gradient sedimentation (Baase and Wang, 1974). No ligase activity could be detected.
Gel electrophoresis and assay quantitation. Reaction mixtures were assayed by a modification of the procedure of Keller and Wendel (1974). The 1% agarose resolving gel was 90 X 125 X 1.5 mm underlain with a 2.5% agarose gel to hold it in place. Both gels contained 0.040 M Tris-acetate (pH 8.4)-0.02 M sodium acetate-O.002 M NazEDTA-0.5 pg/ml of ethidium bromide. Samples (50 ~1 each) were electrophoresed at constant voltage (110 V) for 2.5 hr in the cold room. After electrophoresis, gels were transilluminated with short-wave uv light. (UV Products, San Gabriel, California) and photographed using Polaroid 55 P/N film for 1 min at f4.5 through a Kodak Wratten filter (No. 23A). The negative was scanned on a Joyce-Loebl Mark IIIC microdensitometer. The peak height was found empirically to be directly proportional to the amount of DNA in a given sample provided the sample volume was constant. In addition, however, unreacted
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PM2 DNA was routinely electrophoresed at 100, 50, and 25% of initial concentration as calibration standards. Deviations from linearity occurred at less than 25% initial DNA. The standard deviation of six replicate assays was &a%. Metrizamide density gradients. Aliquots of sonicated nuclei were mixed with metrizamide stock solutions at 4°C to a final volume of 5 ml. Samples were centrifuged at 4°C in a Beckman SW 65 rotor for 48 hr. Fractions (0.25 ml) were collected at 4°C and aliquots were counted for 3H and assayed for enzyme activity. Refractive indices of the fractions were determined at 20°C and corrected to density by a formula given by Rickwood and Birnie (1975). Radioactivity determinations. Aliquots of cell suspensions or homogenates were precipitated in 5% perchloric acid, collected on Millipore HAWP filters, washed twice with 5% HClO+ and hydrolyzed in 1 ml of 5% HClO( in scintillation vials for 40 min at 80°C. Ten milliliters of scintillation cocktail (4 g of PPO-0.3 g of POPOP-667 ml of toluene-333 ml of Triton X-100) was added and radioactivity was measured in a Searle Mark II Liquid Scintillation System. DNA determinations. DNA was measured by a modification of the procedure of Beers and Wittliff (1975). An equal volume of 0.2% SDS was added to the sample and the solution was incubated at 60°C for 10 min. An equal volume of 2 M NaCl was added to the solution and SDS was precipitated at 0°C for 10 min. The precipitate was collected at 2500 g for 5 min and extracted with 1 M NaCl. The combined supernatants were diluted with 1 M NaCl to an appropriate volume. Sample aliquots were adjusted to give a final NaCl concentration of 0.4 M. Equivalent aliquots were made to contain 50 pg/ml of DNase I (Worthington, No. 6330)-5 m&f MgCL. Standards were sea urchin sperm DNA. All samples were incubated at 37°C for 60 min. After chilling on ice, 0.1 vol of 40 pg/ml ethidium bromide was added. Fluorescence was determined on an Aminco-Bowman
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spectrofluorometer with an excitation wavelength of 365 nm and emission wavelength of 592 nm. DNA concentrations were determined after subtacting background of appropriate blanks and taking the difference between the readings without and with DNase digestion and comparing this to the linear standard curve. Protein determination. Samples were precipitated with 20% trichloroacetic acid at 4’C for 1 hr. Precipitates were spun down at 16,000 g for 30 min and washed three times with cold 95% ethanol. The final pellets were dissolved in 0.1% SDS-l M NaOH at room temperature. The samples were analyzed by the procedure of Lowry et al. (1951) using bovine serum albumin as the standard.
VOLIJME 64, 1978
tions of assay, the activity appears to be of the topoisomerase type. In the absence of significant endonuclease activity which would constitute a competing reaction, topoisomerase activity can be quantitated as either the amount of supercoiled DNA lost or the amount relaxed DNA produced. We define 1 unit (U) of activity as the amount of enzyme necessary to convert 50% of the supercoils to relaxed forms measured by the fraction of PM2 supercoils remaining after reaction under our assay conditions. As seen in Fig. 2, the
RESULTS
Characterization
of the Enzyme Activity
A true topoisomerase activity will convert covalently closed circular DNA from a supercoiled to a relaxed form. Sonicates of sea urchin embryo nuclei were tested for such an activity by incubation with purified phage PM2 DNA consisting of 90% supercoils and 10% nicked molecules. The reaction products were separated by ethidium bromide-agarose gel electrophoresis into three species corresponding in order of increasing mobility to nicked, supercoiled, and relaxed forms in the absence of ethidium (Fig. 1). As increasing amounts of nuclear material are added to the reaction mixture, more supercoiled form becomes converted to the relaxed form. The nicked form increases routinely by less than 10% of its original amount (i.e., less than 1% nicking of supercoils). In addition to topoisomerase activity, it is possible to generate relaxed forms from supercoiled forms by a combination of endonuclease and ligase actions to free the closed circular DNA molecules of the topological constraint (Wang, 1971). Aliquots of nuclei were tested for ligase activity under topoisomerase reaction conditions. Since no ligase was detected under condi-
a.
b.
C.
0 DIRECTION OF MIGRATION FIG. 1. Gel electrophoretogram of PM2 DNA reacted with varying amounts of sea urchin blastula nuclear sonicates containing topoisomerase activity. Products of reaction of PM2 DNA with approximately (a) 0, (b) 0.75, and (c) 1.5 U of blastula nuclear enzyme separated on a 1% agarose-ethidium bromide gel. Species from left to right correspond to nicked, supercoiled, and relaxed DNA in the absence of ethidium (Keller and Wendel, 1974).
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NaCl in the incubation is less than 0.05 M or greater than 0.25 M (Fig. 3a). Maximal activity is observed at slightly less than 0.20 M NaCl with a shoulder near 0.10 M. The temperature optimum of the reaction occurs at 30°C with no activity above 50°C (Fig. 3b). Treatment of nuclear sonicates at 100°C for 2 min or with 0.1% sodium dodecyl sulfate completely abolishes activity. The pH optimum centers around neutrality for potassium phosphate buffers (Fig. 3~).
&o.mL E
0
Chromatin Localization 5
IO
AMOUNT ENZYME ADDED t/J) FIG. 2. ation with to reaction lated as a maining.
Proportion of topoisomerase activity variamount of blastula nuclear sonicate added mixture. The amount of activity is calcufraction of the supercoiled DNA peak re-
of the reaction is directly proportional to the amount of nuclear homogenate added up to at least 80% conversion, and there is no significant self-inhibition of the reaction at up to 20 U of excess homogenate added. In an active fraction, the amount of chromatin DNA added for an assay is approximately equal to the amount of supercoiled PM2 DNA reactant. To test for inhibition of the activity by chromatin DNA, increasing amounts of sperm chromatin DNA, which lacks detectable topoisomerase activity (co.5 U/pg), were added to a fixed amount of blastula chromatin (23 U/pg) and the mixtures were assayed. Table 1 shows that there is little inhibition by the sperm chromatin up to a 20-fold DNA excess over PM2 substrate. The sensitivity of the assay is limited by nucleic acids in the homogenates which migrate into the gel at the regions of interest. In practice, however, this is only of concern for sperm nuclei which exhibit no detectable activity and sets an upper limit of activity for these nuclei. Salt, temperature, and pH optima were determined for the nuclear enzyme. No activity is detected if the concentration of
extent
of the Enzyme
Although the activity is detected in nuclear fractions of sea urchin embryos, its presence could be interpreted as a cosedimenting contaminant of the crude nuclei. That this is unlikely is indicated by two types of experiments. First, when our blastula nuclear fraction was purified to chromatin by standard procedures involving salt washes and sedimentation through a 1.7 M sucrose layer, the protein to DNA ratio of the chromatin was one-half that of the nuclei. The ratio of enzyme activity to DNA was only slightly decreased, however (Table 2). Second, if the sonicated blastula nuclei are mixed with metrizamide solutions at low ionic strength (0.05 M Tris-HCl) and centrifuged to equilibrium, the chromatin bands as a single sharp peak at 1.20 g/cc (Fig. 4a), a value previously reported for sheared chromatin (Rickwood and Birnie, TABLE
1
ABSENCEOF~NHIRITION OFTOPOISOMERASE ACTIVITYBYEXCF,SSNLJCLEAR DNA” Sperm nuclear DNA per blastula nuclear DNA 20 6.0 2.0 0.60 0.20 0.00
Percentage supercoiled PM2 DNA remaining 62 70 75 63 70 64
” Varying amounts of sperm nuclei were added to a constant amount of blastula nuclear sonicate and PM2 DNA and the reaction was allowed to proceed. The ratio of blastula nuclear DNA to PM2 DNA in the reaction mixture was 0.80.
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0.0
0.1
02
[NOCII
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40 ‘C
(Ml
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6
7
8
PH
FIG. 3. Enzyme activity optima for blastula nuclear topoisomerase. Reactions were carried out under standard conditions except that (a) NaCl concentration, (b) temperature of incubation, or (c) pH of phosphate buffer was varied. TABLE SPECIFIC
ACTIVITY
Source
Crude nuclei Chromatin
2
OF NUCLEAR ENZYME”
AND CHROMATIN
Units per micro- Micrograms of gram of DNA protein per microgram of DNA 44 38
4.4 2.2
” Crude nuclei made using the saline-EDTA procedure and chromatin were prepared as described under Materials and Methods.
1975). The bulk of the activity (80%) cobands with the bulk of the DNA (90%) in five fractions. No peak of activity is seen at higher densities characteristic of free protein or ribonucleoprotein particles. If the gradient is made 1.0 M in NaCl, however, the activity can be dissociated from the partially deproteinized chromatin (Fig. 4b) and is found at densities characteristic of free protein in metrizamide (Rickwood and Birnie, 1975). These experiments support the proposition that the nuclear enzyme activity is largely bound to chromatin and not a cosedimenting contaminant. We were not able to detect cytoplasmic activity in blastulas after sedimenting nuclei at 10,000 g.
Enzyme Activity During Embryonic Development Enzyme activity per unit DNA was measured for eight-cell, blastula, gastrula, and prism stage nuclei. Enzyme activity per unit protein was measured in whole embryo son-
icates at early cleavage stages, blastula, gastrula, and prism. Since development of the sea urchin up to the prism stage is characterized by a large increase in cell number (and therefore nuclear DNA) but no change in mass (or protein) per embryo, these numbers should be proportional to nuclear activity per cell (or per nucleus) and total activity per embryo respectively. Figure 5 shows a semi-logarithmic plot of cells per embryo, nuclear activity per DNA, or total activity per protein as a function of time of development. Nuclear activity decreases slightly during development, about three-fold from 5 to 72 hr. Total activity increases approximately 400-fold during development, however, roughly parallel to the increase in cell number per embryo. Therefore, it appears that the unfertilized egg contains only a small store of topoisomerase activity (the sperm apparently none) and during embryonic development more activity is produced to keep pace with the increase in the number of cells. The early time points in both graphs show the largest errors but for different reasons. The very low activity per unit protein in whole embryos prior to 10 hr of development is difficult to measure. Interference of endogenous nucleic acid background on the agarose gels limits the amount which can be loaded; at very high inputs of homogenates nuclease action becomes significant. It is clear, however, that a large increase in total activity per embryo
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occurs between early cleavage and blastula. Second, variability is found in eight-cell stage nuclei. This is probably due to isolation from different parts of the cell cycle in cultures from different females (see below).
Enzyme Activity during the Cell Cycle To assay the nuclear activity of the enzyme through the cell cycle, a culture was
0
a
5 FRACTION
IO
15
279
grown in the presence of [3H]thymidine. Every 15 min, two aliquots were taken. One was immediately precipitated in cold 5% perchloric acid for determination of r3H]thymidine incorporation and the second was washed with cold sea water and immediately used for nuclear isolation. The nuclei were resuspended and aliquots were processed for determination of C3H]thymi-
20
NUMBER
Topoisomerase
0 b
5 FRACTION
IO
I5 NUMBER
4. Equilibrium buoyant density gradients of blastula nuclei in metrizamide. [“H]Tbymidine-labeled FIG. blastula nuclei were adjusted to approximately - 40% (w/v) metrizamide solution containing (a) 0.05 MTris-HCI (pH 8) or (b) 1.0 M NaCl-0.05 M Tris-HCl (pH 8) and centrifuged at 50,000 rpm in a SW 65 rotor at 4°C for 48 hr. Aliquots were counted for acid-precipitable radioactivity and assayed for topoisomerase. HATCHING
GASTRULA
HOURS AFTER FERTILIZATION
FIG. 5. Levels of topoisomerase activity and cells per embryo during sea urchin embryonic development. A plot of cells per embryo for S. purpuratus is included, which was taken from Hinegardner (1967). Time of the first three cleavages, hatching, and gastrulation in the cultures was the same as that given by Hinegardner. Total activity is proportional to activity per embryo. Nuclear activity is proportional to activity per nucleus. Each point represents a separate determination.
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dine content or assay of enzyme activity. The profiles of r3H]thymidine incorporation (Fig. 6) in embryos and in nuclei were of essentially identical shape, being two parallel step functions indicating the degree of synchrony in the culture and showing that the DNA synthesis cycle did not progress significantly during the 13-min period during which the nuclear isolation procedure occurred. The amount of thymidine incorporation in the nuclei was 60 to 80% that of whole embryos, reflecting the yield of nuclei during isolation. The enzyme activity per aliquot, which is proportional to activity per embryo, varies in a characteristic cyclic fashion. Activity increases during the S period and reaches a maximum near the end of S. It decreases during Gz and mitosis and reaches a low point near the beginning of S or at telophase of mitosis since these embryos lack a G1 (Hinegardner et al., 1964). The difference in activity between high and low points may be as much as lo-fold per embryo (or 5-fold per unit DNA since DNA doubles by the end of S), and this number
VOLUME 64, 1976
is probably a minimum since the times of isolation might not be at the exact extremes of the cycle and the embryos are not absolutely synchronous. This experiment was repeated twice, and the phasing of the cyclic enzyme change relative to the thymidine incorporation curve was identical, while the amplitude varied. Mixing experiments with the high and low points of the cycle showed additivity of activity, suggesting that neither an inhibitor nor an activator, diffusible and in excess, is present in either preparation. DISCUSSION
The activity detected in isolated sea urchin nuclei appears to be a typical eukaryotic topoisomerase type on the basis of several criteria: (1) relaxation of supercoiled DNA, (2) absence of significant endonuclease, (3) absence of DNA ligase, (4) a salt optimum of 0.2 M, and (5) absence of M$ requirement. The activity is abolished by 0.1% SDS or heating to 100°C for 5 min. The pH optimum for reaction is similar to that reported for the Drosophila enzyme
TIME POST FERTILIZATION hours) FIG. 6. Levels of nuclear topoisomerase activity during cleavage cell cycles. Nuclei were isolated from samples removed at 15-min intervals from a culture grown in the presence of [“Hlthymidine (1 pCi/ml). Equivalent aliquots of the culture and of the isolated nuclei were acid precipitated to determine thymidine incorporation. Equal aliquots of nuclear sonicates were assayed for enzyme activity so that the amount of nuclear activity in this case is proportional to units per embryo rather than units per nucleus as in Fig. 5. Approximate DNA synthetic periods are indicated at the top of the graph. The arrow indicates time of addition of [‘Hlthymidine.
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(Baase and Wang, 1974). The temperature optimum of 30°C and low activity at 37’C may reflect the fact that the sea urchins live and develop at about 15°C. Similar heat sensitivities compared to mammalian enzymes have been reported for sea urchin DNA polymerase (Loeb, 1969) and ribonuclease (Fernlund and Josephsson, 1968). Alternatively, it may reflect a general sensitivity of DNA topoisomerases since the Drosophila activity also shows a temperature optimum near 30°C (Baase and Wang, 1974). The activity present in isolated sea urchin nuclei appears to be fairly tightly bound to the chromatin since it copurifies with the chromatin and bands with chromatin in metrizamide density gradients. Since the nuclei are isolated in the presence of nonionic detergent that strips away most of the nuclear envelope, it is unlikely that the enzyme that we detect is primarily associated with the nuclear membranes (see Rosenberg et al., 1976; Yoshida et al., 1977). The way in which topoisomerase activity is elaborated during sea urchin embryonic development is strikingly different from DNA or RNA polymerase activities. A store of DNA polymerase is present in unfertilized sea urchin egg cytoplasm, and total enzyme activity per embryo remains essentially constant up to gastrula (Fansler and Loeb, 1969; Loeb et al., 1969) while undergoing a gradual shift to the nuclei (Fansler and Loeb, 1972). RNA polymerase II and III activities, which account for the bulk of nuclear RNA synthesis in the early embryo, are also maintained at approximately constant levels per embryo up to gastrulation (Roeder and Rutter, 1970). Other activities associated with DNA synthesis or metabolism remain constant during early development as well, such as DNase (Mazia et al., 1948) and four deoxyribonucleotide kinases (Fansler and Loeb, 1969). In contrast, topoisomerase activity increases per embryo roughly in parallel with the increase in the number of nuclei per
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embryo through the prism stage. We do not know if the increase is due to synthesis or progressive activation of the enzyme. Interestingly, sea urchin RNA polymerase I activity has been reported to increase in proportion to celI number during early development (Roeder and Rutter, 1970). The topoisomerase activity does show at least a lo-fold variation in the nuclei during the cell cycle, increasing during S phase, decreasing during Gz, and reaching a minimum at the end of mitosis. The increase in activity during S probably does not represent a simple cumulative increase in chromatin-associated enzyme due to a duplication of the genes for topoisomerase, since the activity begins to fall immediately after S, although on the average, the proportion of enzyme to nuclear DNA is fairly constant over the early developmental stages up to 72 hr. DNA polymerase shows a cyclic variation in nuclear activity as well but differs from topoisomerase in that it reaches a maximum in the first half of S (Fansler and Loeb, 1972). No correlation is possible with either the overall rate of RNA synthesis or the activity of RNA polymerases through the cell cycle in early sea urchin embryos, since no data have been reported on these subjects. Our results on topoisomerase activity in the cell cycle complement those of Rosenberg et al. (1976), who showed low activities in Go and G1 but a substantial increase in S using extracts of whole cultured cells of WiLZ (human lymphocyte). No Gz or M measurements were made. They suggested that the enzyme might play a role in controlling replication but not transcription because of the low activity in G1. On the other hand, Higashinakagawa et al. (1977) found a topoisomerase enzyme associated with actively transcribing (but not replicating) amplified ribosomal gene chromatin from Xenopus laevis and suggested that it might have a role in transcription. Since a large proportion of protein synthesis in cleaving sea urchin embryos is
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devoted to histones (Kedes, 1976), much of the newly synthesized mRNA accumulation on polysomes is histone mRNA (Nemer et al., 1974), and this accumulation is linked to the S phase of the cell cycle (Kedes and Gross, 1969); the cyclic variation in topoisomerase might be correlated with transcription of histone genes. However, it is not certain that synthesis of histone mRNA sequences represents the bulk of nuclear RNA synthesis in early embryos and recent work with HeLa cells indicates that histone mRNA may be synthesized throughout the cell cycle (Melli et al., 1977). One might expect an enzyme involved in chromosome condensation or decondensation to show a change in activity during mitosis. Our data indicate, however, a steady decline throughout M during which chromosomes are at first condensing and then decondensing. The only nucleus we found totally devoid of activity was the mature sperm nucleus, which is inactive in RNA or DNA synthesis. We are currently attempting to study when and how this nucleus acquires topoisomerase activity in the first cell cycle (which includes a G1 period). We thank Dr. Daniel Mazia for suggesting the ethanol procedure for nuclear isolation and our initial collaboration and Mr. Eric Fairfield for assistance and discussion on the agarose-ethidium gel electrophoretic analyses. This work was supported by grants from the National Institutes of Health HD 09654 to D.L.P. and GM 24544 to J.C.W. REFERENCES BAASF,, W. A., and WANG, J. C. (1974). An w protein from Drosophila melanogaster. Biochemistry 13, 4299-4303. BEEHS, P. C., and WITLIFF, J. L. (1975). Measurement of DNA and RNA in mammary gland homogenates by the ethidium bromide technique. Anal. Biochem. 63,433-441. CHAMPOUX, J. J., and DULFJECCO, R. (1972). An activity from mammalian cells that untwists superhelical DNA-A possible swivel for DNA replication. Proc. Nat. Acad. Sci. USA 69, 143-146. CHAMPOUX, J. J., and MCCONAUGHY, B. J. (1976). Purification and characterization of the DNA untwisting enzyme from rat liver. Biochemistry 15, 4638-4642.
VOLUME
64, 1978
DAVIDSON, E. H. (1976). “Gene Activity in Early Development,” p. 162, Academic Press, New York. ESPEJO, R. T., and CANELO, E. S. (1968). Properties of bacteriophage PM2: A lipid-containing bacterial virus. Virology 34, 738-747. ESPWO, R. T., CANEI,O, E. S., and SINSHEIMER, R. L. (1969). DNA of bacteriophage PM2: A closed circular double-stranded molecule. Proc. Nat. Acad. Sci. USA 63,1164-116&X FANSLER, B. A., and LOEB, L. A. (1969). Sea urchin DNA polymerase: II, Changing patterns of localization during early development. Exp. Cell Res. 57, 305-310. FANSLER, B. A., and LOEB, L. A. (1972). Sea urchin DNA polymerase: IV, Reversible association of DNA polymerase with nuclei during the cell cycle. Exp. Cell Res. 75,433-441. FERNLUND, P., and JOSEPFSSON, L., (1968). Preparation and properties of sea-urchin ribonuclease. Biochim. Biophys. Acta 151,373-382. HIGASHINAKAGAWA, T., WAHN, H., and RF.EDER, R. H. (1977). Isolation of ribosomal gene chromatin. Develop. Biol. 55, 375-386. HINEGARDNER, R. T. (1967). Echinoderms. In “Methods in Developmental Biology” (F. H. Wilt and N. K. Wessells, eds.), pp. 139-155, Cromwell-Collier, New York. HINEGAHDNEH, R. T., RAO, B., and FELDMAN, D. E. (1964). The DNA synthetic period during early development of the sea urchin egg. Exp. Cell Res. 36, 53-61. KEDES, L. H. (1976). Histone messengers and histone genes. Cell 8,321-331. KEDES, L. H., and GROSS, P. R. (1969). Identification in cleaving embryos of three RNA species serving as templates for the synthesis of nuclear proteins. Nature (London) 223, 1335-1339. KF,LLEH, W. (1975). Characterization of purified DNArelaxing enzyme from human tissue culture cells. Proc. Nat. Acad. Sci. USA 72,2550-2554. KELLER, W., and WF,NDEL, I. (1974). Stepwise relaxation of supercoiled SV40 DNA Cold Spring Harbor Symp. Quant. Biol. 39, 199-208. LOEB, L. A. (1969). Purification and properties of deoxyribonucleic acid polymerase from nuclei of sea urchin embryos. J. Biol. Chem. 244, 1672-1681. LOF,B, L. A., FANSIXH, B., WII,LIAMS, R., and MAZIA, D. (1969). Sea urchin nuclear DNA polymerase: I, Localization in nuclei during rapid DNA synthesis. Exp. Cell Res. 57, 298-304. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MAZIA, D., BLUMENTHAL, G., and BENSON, E. (1948). The activity and distribution of DNase and phosphatases in the early development of Arbaciapunctulata. Biol. Bull. 95,250-251. MAZIA, D., PETZELT, R., WILLIAMS, R. O., and MEZA,
POCCIA
ET
AL.
Sea Urchin
I. (1972). A Ca-activated ATPase in the mitotic apparatus of the sea urchin egg (isolated by a new method). Exp. Cell Res. 70,325-332. MELLI, M., SPINELLI, G., and ARNOLD, E. (1977). Synthesis of histone messenger RNA of HeLa cells during the cell cycle. Cell 12, 167-174. NEMER, M., GRAHAM, M., and DUBROFF, L. M. (1974). Co-existence of non-histone messenger RNA species lacking and containing polyadenylic acid in sea urchin embryos. J. Mol. Biol. 89,435-454. PULLEYBANK, D. E., and MORGAN, A. R. (1975). The partial purification of “w” protein from calf thymus. Biochemistry 14.5205-5209. RICKWOOD, D., and BIRNIF., G. D. (1975). Metrizamide, a new density gradient medium. FEBS Lett. 50, 102-110. ROF,DER, R. G., and RUTTEH, W. J. (1970). Multiple RNA polymerases and RNA synthesis during sea urchin development. Biochemistry 9,2543-2553. ROSENBERG, B. H., UNGERS, G., and DEIJTSCH, J. F. (1976). Variation in DNA swivel enzyme activity during the mammalian cell cycle. Nucleic Acids Res. 3.3305-3311.
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VOSBERG, H.-P., GROSSMAN, L. I., and VINOGHAD, J. (1975). Isolation and partial characterization of the relaxation protein from nuclei of cultured mouse and human cells. Eur. J. Biochem. 55,79-93. VOSBEHG, H.-P., And VINOGRAD, J. (1976). Puritication and demonstration of the enzymatic character of the nicking-closing protein from mouse L cells. Biochem. Biophys. Res. Commun. 68,456-464. WANG, J. C. (1971). Interaction between DNA and an Escherichia coli protein w. J. Mol. Biol. 55, 523-533. WANG, J. C. (1973). Protein w: A DNA swivelase from E. coZi?Zn “DNA Synthesis in Vitro.” (R. D. Wells and R. B. Inman, eds.), pp. 163-174. University Park Press, Baltimore, Md. WANG, J. C., and LIU, L. F. (1978). DNA topoisomerases: Enzymes which catalyze the concerted breaking and rejoining of DNA backbone bonds. In “Molecular Genetics, Part 3” (H. Taylor, ed.), Acad. Press, New York. in press. YOSHIDA, S., UNC.ERS, G., and ROSENBERG, B. H. (1977). DNA swivel enzyme activity in a nuclear membrane fraction. Nucleic Acids. Res. 4, 223-228.
