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Subcellular localization of DNA polymerase γ and changes in its activity in sea urchin embryos
Comp. Biochem. PhysioL Vol. 91B, No. 3, pp. 525-530, 1988
0305-0491/88 $3.00+0.00 © 1988 Pergamon Press plc
Printed in Great Britain
SUBCELLULAR LOCALIZATION OF D N A POLYMERASE A N D CHANGES IN ITS ACTIVITY IN SEA URCHIN EMBRYOS MASAKISHIODA Department of Physiological Chemistry and Nutrition, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113, Japan (Received 8 December 1987)
Abstract--l. Subeellular localization and changes in the activity of DNA polymerase y were examined in sea urchin eggs and embryos. 2. The enzyme was shown to be localized predominantly in mitochondria by differential and isopycnic centrifugation. 3. During embryogenesis, the enzyme activity per embryo remained constant until blastula stage, and thereafter increased. 4. Similarly mitochondrial DNA per embryo increased, indicating that mitochondrial DNA replication starts during embryogenesis. 5. The 7-activity per mitochondrial DNA remained constant during embryogenesis. 6. These results suggest that mitochondria contain a constant amount of replicative enzyme (DNA polymerase Y) regardless of mitochondrial DNA replication, which differs from the case of nuclear DNA replication.
INTRODUCTION
Three species of D N A polymerase (~,/~ and ~) have been found and classified in eukaryotes (H/ibscher, 1983). D N A polymerases • and 7 are replicative enzymes, whereas D N A polymerase/~ may be related to D N A repair (Hiibscher, 1983). D N A polymerase c~is essential for nuclear D N A replication (Hiibscher, 1983). In general, the enzyme is localized in the nucleus (Weissbach, 1977) and its activity changes with nuclear D N A replication during cell cycle or cell proliferation (Chang and Bollum, 1972; Chiu and Baril, 1975), except in the case of animal eggs and embryos (Zierler et al., 1985). On the other hand, D N A polymerase ~ participates in mitochondrial D N A replication (Hfibscher, 1983). Mitochondria contain only the polymerase, but the enzyme was not always found in only mitochondria: large portions of the enzymes were detected in the soluble fraction in rat liver and HeLa cells (Bolden et al., 1977) and some of the enzymes were recovered in the nucleus in chick embryos and rat brain (Bertazzoni et al., 1977; Hiibscher et al., 1977). Thus, further studies are required to elucidate the subcellular localization of D N A polymerase ~,. It is not well known whether the enzyme activity changes with mitochondrial D N A replication or not. During animal embryogenesis, it was found that a large amount of D N A polymerase c¢ accumulates in the cytoplasm of unfertilized eggs (Fansler and Loeb, 1969; Margulies and Chargaff, 1973; Shioda et al., 1977; Nagano et al., 1982). The enzyme migrates to the nucleus to participate in nuclear, D N A replication, which oocurs at an extremely rapid rate after fertilization (Fansler and Loeb, 1969; Loeb and Fansler, 1970; Shioda et al., 1980; 'Shioda and Nagano, 1983). As for D N A polymerase y, Fox et fd.
(1980) have reported that almost all the enzyme is localized in mitochondria in Xenopus eggs, and that the level of the activity per embryo remains constant throughout early embryogenesis. Similar results were obtained for teleost fish embryos (Mikhailov and Gulyamov, 1983). On the contrary, in sea urchin embryos, Gadaleta et al. (1977) have reported that mitochondrial D N A polymerase activity remains constant until the early blastula stage and then it rises, reaching a maximum at the time of hatching; thereafter it decreases to 50% of the maximum level, remaining constant up to the gastrula stage. Habara et al. (1979) found D N A polymerase 7 in the sea urchin nuclear fraction, although subcellular localization of the enzyme was not characterized. Thus, different viewpoints on behaviour of D N A polymerase ~: during embryogenesis were drawn in frog and sea urchin embryos. In the present study, we examined subcellular localization of D N A polymerase ~, and changes in its activity during embryogenesis in relation to mitochondrial D N A replication, in sea urchin eggs and embryos. The results revealed that the enzyme is localized predominantly in mitochondria and that the enzyme activity per embryo increases during embryogenesis, whereas the activity per mitochondrial D N A is kept constant. Difference in behaviour of the enzyme between frog and sea urchin embryos is also discussed. MATERIALS AND METHODS
Eggs and embryos Gametes of the sea urchin, Hemicentrotus pulcherrimus, were obtained by injection of 0.5 M KCI into the body cavity. Fertilized eggs (2 × 104ernbryos/ml) were allowed to develop at 20°C for the indicated time with gentle stirring
525
526
MASAKI SHIODA
in artificial sea-water (Jamarin Laboratory, Osaka, Japan). After washing three times each with artificial sea-water and 1.0 M glucose, the eggs or embryos were used immediately or stored at -80°C, the results being identical.
Chemicals Unlabelled deoxyribonucleoside 5'-triphosphates (dATP, dCTP, dGTP and dTTP) and [3H]dTTP (96.8 Ci/mM) were purchased from Boehringer-Mannheim (Mannheim, FRG) and New England Nuclear (Boston, Massachusetts, USA), respectively. Calf thymus DNA was obtained from Sigma (St Louis, Missouri, USA) and activated according to the method of Fansler and Loeb (1974). N-Ethymaleimide and poly(rA)'oligo(dT)m 0 were from Wako (Osaka, Japan) and Pharmacia (Uppsala, Sweden), respectively. Pancreatic RNase (RNase A) was obtained from BoehringerMannheim, and treated at 100°C for~ 15 min before use, Cell fractionation by differential or isopycnic centrifugation For cell fractionation by differential centrifugation, eggs (1.0 × 105 per ml) were homogenized in 1.0ml of a 1.2M sucrose solution containing 5 mM 2-mercaptoethanol and 5 mM MgC12. The homogenate was centrifuged at 1000g for 5 min. The supernatant was again centrifuged at 10,000 g for 30min, followed by re-centrifugation of the 10,000g supernatant at 100,000g for 90min. For each centrifugation, 2.0 M sucrose was layered on the bottom of the centrifuge tube as a cushion. Aliquots (2-10 # 1) of the supernatants or precipitates suspended in 200gl of a 0.25M sucrose solution containing 5 mM 2-mercaptoethanol and 5 mM MgC12 were assayed for DNA polymerase activities. For cell fractionation by iospycnic centrifugation, eggs (1.0 x l0 s per ml) were homogenized in 1.0 ml of a 0.25 M sucrose solution containing 5 mM 2-mercaptoethanol and 5 mM MgCI 2. The homogenate (0.3 ml) was applied onto a 4.7ml 1(L70% (w/v) linear sucrose gradient containing 5 mM 2-mercaptoethanol and 5 mM MgCI2, followed by isopycnical centrifugation at 55,000 rpm for 5 hr at 2°C, in a Hitachi swinging-bucket rotor, RPS 55T. After the centrifugation, fractions (0.13 ml) were collected from the bottom of the centrifuge tube, The buoyant density of each fraction was determined by measuring the refractive index. Aliquots (5/~1) of the fractions were assayed for DNA polymerase activities. Assay of DNA polymerases ct and [3 The activities of DNA polymerases ~t and fl were determined from the incorporation of [3H]dTMP into the acid-insoluble materials. The standard reaction mixture contained 50 mM Tris-maleate buffer (pH 8.0), 7 mM MgC12, 20/~M each of dATP, dCTP and dGTP, 10/~M dTTP containing 0.4/zCi [3H]dTTP (400 cpm = 1 pmol dTTP), 40 mM NaCI, 2.5/~g activated DNA, 10#g bovine serum albumin and 10% glycerol in a final volume of 25/zl. Incubation was carried out in the absence and presence of 20 mM N-ethylmaleimide for 30 min at 26°C, and DNA polymerase ~t and fl activities were determined as the drug-sensitive and drugresistant ones, respectively, as described previously (Usuki and Shioda, 1986). Assay of DNA polymerase 7 Before the assays, the enzyme solutions were treated ~vith 50 mM Tris-HC1 (pH 8.0), 1.0% Triton'X-100, 250 mM NaCI, l mM spermidine, 1 mg/ml bovine Serum albumin and 5 mM EDTA (final concentrations) at 0°C for 10 mih. Incubation was carried out for 30 min at 26°C in a reaction mixture (25#1) containing 50mM Tris-maleate buffer (pH 8.5), 5 mM MgC12, 100mM NaC1, 20 mM potassium phosphate (pH 7.9), 1.0#M dTTP containing 1.0/~Ci [3H]dTTP (10,000cpm = 1 pmol dTTP), 0.5#g poly(rA). oligo(dT)10, I0 #g bovine serum albumin and 10% glycerol (final concentrations). After the incubation, 25/~1 of a carrier solution containing 200mM Tris-HC1 (pH 8.0),
1.0% sodium dodecyl sulphate, 0.5M NaCI, 400mM EDTA, 100#M each of dATP, dCTP, dGTP and dTTP, and 4 mM ATP was added to stop the reaction and to reduce nonspecific binding of the radioactivity to the filter paper. The radioactivity incorporated into the acidinsoluble materials was determined as described previously (Shi0da et al., 1980). To assure the ?-polymerase activity, incubations were carried out in the absence and presence of 20 mM N-ethylmaleimide, and DNA polymerase ? activity was determined as the drug-sensitive activity.
Measurement of DNA, RNA and protein contents In each fraction prepared by isopycnic centrifugation as described above, nucleic acids were extracted as described previously (Dillon et al., 1985), and RNA content was determined by the method of Mejbaum (1939). The fractions of nucleic acids in 50mM Tris-HCl (pH 7.5) and 5mM MgCI 2 were treated with pancreatic RNase (0.05 mg/ml) at 37°C for 60 min, followed by NaOH treatment (0.3N) at 37°C for 17hr. After neutralization with 1.0 N HC1, DNA was extracted as described (Dillon et al., 1985), and the content of DNA was determined by the method of Burton (1956). To determine the content of mitochondrial DNA, the fractions prepared by the isopycnic centrifugation were treated with EDTA (5 mM), stood for 10 hr at 4°C and then centrifuged at 10,000g for 30min. The precipitate was dissolved in 50 #1 of 1.0% sodium dodecyl sulphate, 50 mM Tris-acetate (pH 8.0), 2 mM EDTA and 15 mM NaC1, and then subjected to 0.7% neutral agarose gel electrophoresis as described previously (Shioda, 1986a). The amount of mitochondrial DNA in the gel was determined by comparison with that of standard DNA, using a densitometer. As the standard DNA, mitochondrial DNA was isolated from unfertilized eggs according to the method of Kaneko and Terayama (1975). Protein content was determined by the method of Lowry et al. (1951). RESULTS
Subcellular distribution of D N A polymerase 7 analyzed by differential centrifugation To examine the subcellular distribution of D N A polymerase 7 in the eggs, the egg h o m o g e n a t e was fractionated by differential centrifugation. In this experiment, D N A polymerase a activity was also examined as a control, since subcellular distribution of the enzyme has been characterized in sea urchin eggs (Shioda et al., 1980). Non-buffered m e d i u m was used to avoid leakage of enzymes from the organelle in which the enzymes were originally localized, as reported for D N A polymerase a (Lynch et al., 1975), a l t h o u g h the separation between different kinds of organelle became poor. W h e n a non-buffered 0.25 M sucrose solution was used, considerable a m o u n t s of D N A polymerase ~ activity were recovered in the 1000 g a n d 10,000 g precipitates, a n d the yield of the activity was poor. These results differ from our previous one, i.e. almost all the D N A polymerase was f o u n d to be localized on the r o u g h endoplasmic reticulum (Shioda et al., 1980), suggesting that cosedimentation of organella occurred t h r o u g h nonspecific binding between organella u n d e r the condition used. W h e n a non-buffered 1.2 M sucrose solution was used to prevent yolk sedimentation, 7 7 % of the D N A polymerase a was recovered in the 100,000 g precipitate (Table 1), the results thus being similar to the previous ones. This suggests t h a t
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D N A p o l y m e r a s e ), in sea urchin e m b r y o s Table 1. Subcellular distribution of DNA polymerase 7 on fractionation by differential centrifugation
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subcellular fractionation is available to determine the localization of DNA polymerase y under these conditions. Under these conditions, 80% of the DNA polymerase ~ activity was recovered in the 10,000g precipitate (Table 1), indicating that DNA polymerase y is localized predominantly in mitochondria.
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Unfertilized eggs were homogenized in 40% sucrose, 5 mM 2-mercaptoethanol and MgCI 2, and then fractionated by differential centrifugation, followed by assaying of DNA polymerases = and ),, as described in the text.
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Fig. 1. Subcellular d i s t r i b u t i o n o f D N A polymerases, as
analyzed by isopycnic centrifugation. Unfertilized eggs were homogenized in a non-buffered solution, followed by isopycnic centrifugation, and then assaying of DNA polymerases in each fraction, as described under Materials and Methods. (©) DNA polymerase ct activity; (A) DNA polymerase fl activity; (0) DNA polymerase ~, activity; (---), absorbance at 280 nm; (x) buoyant density.
On isopycnic centrifugation with a non-buffered solution, two major peaks (a and b in Fig. 1) of 2.5 mg in peak (b). Based on this value, the purity of absorbance at 280 nm were obtained. The activity mitochondria in peak (b) was estimated to be 78%, profiles of DNA polymerases ct and ~ coincided with assuming that the ratio, protein content (mg) to DNA peak (a). Peak (a) seemed to correspond to the rough content (~g), of pure mitochondria is 1.96, as reendoplasmic reticulum fraction, judging from its ported for Xenopus egg mitochondria (Dawid, 1966). buoyant density (mean _ SD: 1.262 __+0.020 g/cm 3, These results indicate that peak (b) is the main N = 8) and content of rRNA (0.13 mg/mg protein), mitochondrial fraction. Thus, DNA polymerase ~ in as characterized previously (Shioda et al., 1980). On peak (b) is indicated to be localized in mitochondria, the other hand, distribution of DNA polymerase 7 since the buoyant densities of the enzyme and mitoactivity coincided with peak (b). The buoyant density chondria were same. DNA polymerase 7 activities per of the peak was 1.216___0.012 (mean +__SD) g/cm 3 protein content were different from each other in (N = 8), in agreement with that of mitochondria in peak (a), peak (b), fraction (c) and fraction (d), while rat liver (Beaufay et al., 1959). These results suggest the enzyme activity per mitochondrial DNA of peak that DNA polymerase y is localized in mitochondria. (b) was similar to that of peak (a) and fraction (c), but To quantify the localization of DNA polymerase differed from that of fraction (d). This suggests that in mitochondria, fractions prepared by the isopycnic DNA polymerase ~ is localized in the same kind of centrifugation in Fig. 1 were grouped tentatively into organelle (mitochondria) in peak (a), peak (b) and five fractions: bottom (precipitate), peak (a) (fraction fraction (c), but not in fraction (d). Peak (a), peak (b) number 1-9), peak (b) (fraction number 10-20), (c) and fraction (c) contained 14%, 72% and 8% of (fraction number 21-29) and (d) (fraction number DNA polymerase 7 activity. Thus, it is suggested that 30-40), and DNA and protein contents of each at least 96% of DNA polymerase ~, is localized in fraction were determined (Table 2). Peak (b) con- mitochondria. tained 73% of mitochondrial DNA and little nuclear On the isopycnic centrifugation of the homogenate DNA, whereas the bottom fraction:contained 83% of o f 15hr old embryos (late blastulae), 9%, 15%, nuclear DNA and little mitochondrial DNA. The 74% and 7% of DNA polymerase 7 activity were protein content per /zg mitochondrial DNA was recovered in the nuclear fraction (buoyant density Table 2. Characterization of each fraction prepared by isopycnic centrifugation DNA content
Fraction Bottom Peak (a) Peak (b) (c) (d)
DNA polymerase 7-activity pmol (%) 0 2.5 13.0 1.4 1.1
(0) (14) (72) (8) (6)
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mit DNA #g (%) 0 0.6 2.4 0.3 <0.1
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Unfertilized eggs were fractionated by the isopycnic centrifugation as described in Fig. 1. Fractions corresponding to those in Fig. 1 were grouped to five fractions: bottom (corresponding to precipitate), peak (a) (fraction number 4-9), peak (b) (fraction number 10-20), (c) (fraction number 21-30) and (d) (fraction number 31-40). The grouped fractions were characterized. Nuclear DNA content was determined by subtracting the content of mitochondrial DNA from total DNA content determined by the method of Burton (1956). Each value corresponds to that from 1.0 x 106 eggs. mitDNA represents mitochnndrial DNA.
528
M A S A K I SI'IIODA
1.33 g/cm 3) (Shioda et al., 1982), rough endoplasmic reticulum fraction, mitochondrial fraction and other fraction (buoyant density less than 1.18g/cm3), respectively.
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of the buffered solution. These results suggest that almost all the DNA polymerase y leaked from the mitochondria into the soluble fraction in the presence of Tris buffer. Figure 2B shows the relationship between the extent of leakage of DNA polymerase y from mitochondria and the concentration of Tris-HC1 buffer (pH 8.0); it shows that half the enzyme leaked out in the presence of less than 5 mM Tris buffer and that the leakage occurred in a concentration-dependent manner. Almost all of the enzyme also leaked out in the presence of 0.1 M NaCI or KCI without Tris buffer. On the other hand, almost all the DNA polymerase ~t and about 30% of the DNA polymerase fl remained on the endoplasmic reticulum in the presence of 20 mM Tris-HC1 buffer (Fig. 2A). Almost all of both polymerases were free from organella in the presence of 0.1 M NaCl or KCl without Tris buffer.
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Changes in DNA polymerase y activity and the amount of mitochondrial DNA during embryogenesis
Leakage of DNA polymerase Y from mitochondria When the egg homogenate was prepared in the presence of a buffered solution (20 mM Tris-HCl, pH 8.0), almost all the DNA polymerase ? activity was recovered in fractions with densities lower than that of mitochondria, and the activity continued to sediment (Fig. 2A). The peak (b) material equilibrated at a Similar density to that in the absence o
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Mitochondrial fractions were prepared from 0 hr old (unfertilized eggs), 5 hr old (morulae), l0 hr old (early blastulae), 15 hr old (late blastulae), 20 hr old (gastrulae) and 25 hr old (late gastrulae) embryos by isopycnic centrifugation under the non-buffered conditions, and then the DNA polymerase Yactivity and the amount of mitochondrial DNA in each fraction determined (Fig. 3). DNA polymerase V activity per embryo remained almost constant during the first
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Fig. 2. Effect of Tris buffer on the leakage of DNA polymerase 7 from mitochondria. (A) Subcellular distribution of DNA polymerase 7 in the presence of Tris buffer on isopycnic centrifugation. Unfertilized eggs were homogenized in 0.25 M sucrose, 20 mM Tris-HCl (pH 8.0), 5 mM 2-mercaptoethanol and 5 mM MgCI2, followed by isopycnical centrifugation, as described under Materials and Methods, except that the sucrose gradient contained 20 mM Tris-HCl (pH 8.0), and then assaying of DNA polymerases. (O) DNA polymerase ~t activity; (A) DNA polymerase fl activity; (O) DNA polymerase Vactivity; ( - - - ) absorbance at 280 nm; (x) buoyant density. (B) Relationship between the Tris buffer concentration and the leakage of DNA polymerase )' from mitochondria. The mitochondrial fraction was prepared from unfertilizedeggs by isopycniccentrifugation, as described under Materials and Methods. The fraction suspended in 200pl of 0.25 M sucrose, 5 mM 2-mercaptoethanol and 2 mM EDTA was treated with the indicated concentrations of Tris-HC1 (pH 8.0), and then applied on 0.7 ml of a 1.2 M sucrose solution containing 5 mM 2-mercaptoethanol with a 2.0 M sucrose cushion, followed by centrifugation at 10,000g for 30 rain. The precipitate on the cushion was suspended in 20-30 pl of 0.25 M sucrose and 5 mM 2-mercaptoethanol, and then aliquots of the fractions obtained were assayed for DNA polymerase )'. The figure shows the activity remaining in the mitochondria. 100% corresponds to 0.3 pmol dTMP incorporated.
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Fig. 3. Changes in DNA polymerase y activity (A), the amount of mitochondrial DNA (B) and the specific activity of DNA polymerase 7 (C) during embryogenesis. Embryos were developed at 20°C. At 10 and 20 hr after fertilization, hatching and gastrulation occurred, respectively. From embryos at the indicated stages, mitochondrial fractions were prepared by isopycnic centrifugation, and then DNA polymerase 7 activity and the amount of mitochondrial DNA were determined, as described under Materials and Methods. The specific activity of DNA polymerase 7 is expressed as the ratio of the ),-activity (nmol dTMP incorporated) to the amount of mitochondrial DNA (mg). Each point and bar represents the mean + SD for three separate experiments.
DNA polymerase ~, in sea urchin embryos 10 hr after fertilization. But, thereafter, it began to increase gradually, reaching about 3-fold the level in eggs by 25 hr after fertilization (Fig. 3A). On the other hand, the amount of mitochondrial DNA per embryo was found to be increased 5 to 10 hr after fertilization, and thereafter continued to increase. By 25 hr after fertilization, it had increased to about 3-fold the level in eggs (Fig. 3B). The specific activity of DNA polymerase y (DNA polymerase activity per mitochondrial DNA) showed a slight decrease at about 10hr after fertilization, but, thereafter, returned to a similar level as that in eggs (Fig. 3C). DISCUSSION Although the subcellular distribution of DNA polymerases changed with the conditions used, the bulk of DNA polymerase 7 was recovered in the mitochondrial fraction on the differential and isopycnic centrifugation under a certain condition by which DNA polymerase ~ was distributed in the rough endoplasmic reticulum (Tables 1 and 2, Fig. 1). These results exclude the possibility of a non-specific distribution of DNA polymerase 7 caused by the low ionic conditions. Therefore, we conclude that DNA polymerase 7 is localized predominantly in mitochondria in sea urchin eggs and embryos. A similar conclusion was drawn for Xenopus eggs (Fox et al., 1980) and some mammalian cells (Tanaka and Koike, 1978; Bazzicalupo, 1979). Some studiesr suggested that some portion of DNA polymerdse ~, is localized in the nucleus (Bertazzoni et al., '1977; Hiibschcr et al., 1977; Kalf et al., 1980). In the I~resent study, a small amount of the enzyme activity was also detected in the nuclear fraction of embryos, but it Was uncertain whither the activity originated from nuclei or from mitochondria. To elucidate this localization, further studies are required. Lynch et al. (1975) showed that DNA polymerase leaks easily from nuclei with Tris buffer or KC1. Similarly, the present study indicated that DNA polymerase ), leaks from mitochondria in the presence of Tris buffer (Fig. 2). Although it is not known whether or not the leakage of DNA polymerase ), occurs to the same extent in other cells, the leakage may explain the low recovery of DNA polymerase y in the mitochondrial fraction and localization of the enzyme in the soluble fraction, reported for some cells (Bolden et al., 1977). Many contradictory results have been reported as to the proliferation of mitochondria during sea urchin embryogenesis: some indicated that mitochondrial proliferation does not occur in unfertilized eggs or embryos up to the pluteus stage (Matsumoto et al., 1974; Bresch, 1978; Rinaldi et al., 1979), but others showed that it occurs after fertilization (Shaver, 1956; Anderson, 1969; Bresch, 1973; Kaneko and Terayama, 1975). In the present study, although the exact time the increase started was uncertain, the mitochondrial DNA content per embryo was found to be increased 5 to 10 hr after fertilization (Fig. 3B). DNA polymerase 7 activity also began to increase 15 hr after fertilization (Fig. 3A). Thus, the specific activity of DNA polymerase y remained almost constant throughout embryogenesis, with a slight decrease
529
at the early blastula stage (Fig. 3C). This may imply the following: pre-existing DNA polymerase ), participates in mitochondrial DNA replication at the early blastula stage; after the replication, the doubling of mitochondria including DNA polymerase ~ occurs; thereafter, mitochondria begin to proliferate asynchronously. An essentially similar relationship has been reported for mitochondriai DNA replication and a mitochondrial enzyme, succinateeytochrome-c reductase, in embryos of this sea urchin species (Kaneko and Terayama, 1975). In Xenopus (Fox et aL, 1980) and teleost fish (Mikhailov and Gulyamov, 1983), DNA polymerase ), activity per embryo was shown to remain constant during early embryogenesis, although it increased in the present study. In the former species of embryos, the amount of mitochondria also remains constant during the stage, but it begins to increase at the later stage. The difference in the amount of the enzyme activity between the former species of embryos and the present embryos may be due to the timing of mitochondrial proliferation. Thus, the present results are comparable, rather than contradictory, to those reported for Xenopus and teleost fish embryos as to the enzyme activity per mitochondrion. Gadeleta et al. (1977) have reported on the mitochondrial DNA polymerase in sea urchin embryos. However, under the conditions they used, a considerable amount of DNA polymerase 7 is supposed to have leaked, judging from the present results (Table 2), since mitochondria were prepared in the presence of 100 mM Tris-HCl with 240 mM KC1 in their study. So, the DNA polymerase activity they reported does not seem to represent all of DNA polymerase ~ in mitochondria in vivo, although their results may reflect some physiological aspects of mitochondrial DNA replication. The specific activity of DNA polymerase remained almost constant regardless of whether mitochondrial proliferation occurred or not during embryogenesis (Fig. 3C). Similar results have been obtained for teleost fish (Mikhailov and Gulyamov, 1983). The enzyme was found in sea urchin sperm, in which mitochondrial DNA replication does not occur (Habara et al., 1980). These results indicate that mitochondrial DNA replication is not regulated by the level of DNA polymerase ), activity, although the polymerase is essential for mitochondrial DNA replication. On the other hand, the specific activity of DNA polymerase ~t changes with nuclear DNA replication during the cell cycle and cell proliferation (Usuki and Shioda, 1986). Therefore, it is suggested that different regulatory mechanisms are involved in mitochondrial and nuclear DNA replication. Acknowledgements--This work was supported by Grantsin-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan. REFERENCES
Anderson W. A. (1969) Nuclear and cytoplasmic DNA synthesis during early embryogenesis of Paracentrotus lividus. J. Ultrastruct. Res. 26, 95-110. Bazzicalupo P. (1979) Mitochondrial DNA polymerase from Xenopus laevis oocytes. Biochem. biophys. Res. Commun. 87, 1218-1225.
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MASAKISHIODA
Beaufay H., Bendall D. S., Baudhuin P., Wattiaux R. and de Duve C. (1959) Tissue fractionation studies. Biochem. J. 73, 628-637. Bertazzoni U., Scovassi A. I. and Brun G. M. (1977) Chick embryo DNA polymerase 7. Eur. J. Biochem. 81, 237-248. Bolden A., Noy G. P. and Weissbach A. (1977) DNA polymerase of mitochondria is a 7-polymerase. J. biol. Chem. 252, 3351-3356. Bresch H. (1973) Mitochondrial DNA metabolism in young Psammechinus miliaris embryos. FEBS Lett. 31, 233-236. Bresch H. (1978) Mitochondrial profile densities and areas in different developmental stages of the sea urchin Sphaerechinus granularis. Expl. Cell Res. 111, 205-209. Burton K. (1956) A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62, 315-323. Chang L. S. M. and Bollum F. J. (1972) Variation of deoxyribonucleic acid polymerase activities during rat liver regeneration. J. biol. Chem. 247, 7948 7950. Chiu R. W. and Baril E. F. (1975) Nuclear DNA polymerases and the HeLa cell cycle. J. biol. Chem. 250, 7951-7957. Dawid I. B. (1966) Evidence for the mitochondrial origin of frog egg cytoplasmic DNA. Proc. nam. Acad~ Sci. U.S.A. 56, 269-276. Dillon J. R., Bezarson G. S. and Yeung :K, (1985) Recombinant DNA Methodology (Edited b'y Dillon J. R., Nasim A. and Nestman E. R.), pp. 1-126. John Wiley and Sons, New York. Fansler B. and Loeb L. A. (1969) Sea urchin nuclear DNA polymerase. Expl Cell Res. 57, 305-310. Fansler B. S. and Loeb L. A. (1974) Sea urchin DNA polymerase. Meth. Enzym. 29, 53-70. Fox A. M., Breaux C. B. and Benbow R. M. (1980) Intracellular localization of DNA polymerase activities within large oocytes of the frog, Xenopus laevis. Devl Biol. 80, 79-95. Gadaleta M. N., Nicotra A., Del Prete M. G. and Saccone C. (1977) DNA polymerase activity in isolated mitochondria of Paracentrotus lividus at various stages of development. Cell D/ft. 6, 85-94. Habara A., Nagano H. and Mano Y. (1979) Identification of 7-like DNA polymerase from sea urchin embryos. Biochim. biophys. Acta 561, 17-28. Habara A., Nagano H. and Mano Y. (1980) Characterization of DNA polymerases in mature sperm of the sea urchin. Biochim. biophys. Aeta 608, 287-294. Hiibscher U., Kuenzle C. C. and Spadari S. (1977) Identity of DNA polymerase 7 from synaptosomal mitochondria and rat brain nuclei. Eur. J. Biochem. 81, 249-258. Hiibscher U. (1983) DNA polymerases in prokaryotes and eukaryotes. Experientia 39, 1-126. Kalf G. F., Maguire R. F., Metrione R. M. and Koszalka T. R. (1980) DNA replication by isolated rat trophoblast nuclei. Devl Biol. 77, 253-270. Kaneko T. and Terayama H. (1975) The behavior of mitochondrial DNA and enzyme during the course of the early development of sea urchin, Hemicentrotus pulcherrimus. J. Fac. Sci. Tokyo Univ., Seet. IV, 13, 285-297. Loeb L. A. and Fansler B. (1970) Intracellular migration of DNA polymerase in early developing sea urchin embryos. Biochim. biophys. Acta 217, 50-55.
Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 256-275. Lynch W. E., Surry S. and Lieberman I. (1975) Nuclear deoxyribonucleic acid polymerases of liver. J. biol. Chem. 250, 8179-8183. Matsumoto L., Kasamatsu H., Pik6 L. and Vinograd J. (1974) Mitochondrial DNA replication in sea urchin oocytes. J. Cell Biol. 63, 140-159. Margulies L. and Chargaff E. (1973) Survey of DNA polymerase activity during the early development of Drosophila melanogaster. Proc. nam. Acad. Sci. U.S.A. 70, 2946--2950. Mejbaum W. (1939) Ober die Bestimmung kleiner Pentosemengen, insbesondere in Derivaten der Adenyls/iure. Z. Physiol. Chem. 258, 117-120. Mikhailov V. S. and Gulyamov D. B. (1983) Changes in DNA polymerase ~, fl, 7 activities during early development of the teleost fish Misgurnus fossilis (loach). Eur. J. Biochem. 135, 303-306. Nagano H., Okano K., Ikegami S. and Katagiri C. (1982) Changes in intracellular location of DNA polymerase during oocyte maturation of the toad, Bufo bufo japonicus. Biochem. biophys. Res. Commun. 1116, 683-690. Rinaldi A. M., De Leo G., Arzone A., Salcher I., Storace A. and Mutolo V. (1979) Biochemical and electron microscopic evidence that cell nucleus negatively controls mitochondrial genomic activity in early sea urchin development. Proc. natn. Acad. Sci. U.S.A. 76, 1916-1920. Shaver J. R. (1956) Mitochondrial populations during development of the sea urchin. Expl Cell Res. 11, 548-559. Shioda M., Nagano H. and Mano Y. (1977) Cytoplasmic location of DNA polymerase ~ and fl of sea urchin eggs. Biochem. biophys. Res. Commun. 78, 1362-1368. Shioda M., Nagano H. and Mano Y. (1980) Association of DNA polymerase ~ and/~ with rough endoplasmic reticulum in sea urchin eggs and changes in subcellular distribution during early embryogenesis. Eur. J. Biochem. 108, 345-355. Shioda M., Nagano H. and Mano Y. (1982) Transition of DNA polymerase ~ and endoplasmic reticulum during gastrulation of the sea urchin. Devl Biol. 91, 111-120. Shioda M. and Nagano Y. (1983) Localization of DNA polymerase ~ on the nuclear membrane in sea urchin embryos. Expl Cell Res. 146, 349-360. Shioda M. (1986a) DNA synthesis in vitro with an endoplasmic reticulum DNA polymerase complex from unfertilized sea urchin eggs. Eur. J. Biochem. 160, 571-578. Shioda M. (1986b) Two forms of DNA polymerase ~ in sea urchin eggs and embryos. Zool. Sci. 3, 555-558. Tanaka S. and Koike K. (1978) DNA polymerase V is localized in mitochondria. Biochem. biophys. Res. Commun. 81, 791-797. Usuki S. and Shioda M. (1986) Increase in DNA polymerase activity associated with DNA synthesis due to FSH or oestrogen in ovaries of immature rats. J. Endocr. 110, 353 360. Weissbach A. (1977) Eukaryotic DNA polymerases. A. Rev. Biochem. 46, 25~,7. Zierler M. K., Marini N. J., Stowers D. J. and Benbow R. M. (1985) Stockpiling of DNA polymerases during oogenesis and embryogenesis in the frog, Xenopus laevis. J. biol. Chem. 260, 974-981.
0305-0491/88 $3.00+0.00 © 1988 Pergamon Press plc
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SUBCELLULAR LOCALIZATION OF D N A POLYMERASE A N D CHANGES IN ITS ACTIVITY IN SEA URCHIN EMBRYOS MASAKISHIODA Department of Physiological Chemistry and Nutrition, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113, Japan (Received 8 December 1987)
Abstract--l. Subeellular localization and changes in the activity of DNA polymerase y were examined in sea urchin eggs and embryos. 2. The enzyme was shown to be localized predominantly in mitochondria by differential and isopycnic centrifugation. 3. During embryogenesis, the enzyme activity per embryo remained constant until blastula stage, and thereafter increased. 4. Similarly mitochondrial DNA per embryo increased, indicating that mitochondrial DNA replication starts during embryogenesis. 5. The 7-activity per mitochondrial DNA remained constant during embryogenesis. 6. These results suggest that mitochondria contain a constant amount of replicative enzyme (DNA polymerase Y) regardless of mitochondrial DNA replication, which differs from the case of nuclear DNA replication.
INTRODUCTION
Three species of D N A polymerase (~,/~ and ~) have been found and classified in eukaryotes (H/ibscher, 1983). D N A polymerases • and 7 are replicative enzymes, whereas D N A polymerase/~ may be related to D N A repair (Hiibscher, 1983). D N A polymerase c~is essential for nuclear D N A replication (Hiibscher, 1983). In general, the enzyme is localized in the nucleus (Weissbach, 1977) and its activity changes with nuclear D N A replication during cell cycle or cell proliferation (Chang and Bollum, 1972; Chiu and Baril, 1975), except in the case of animal eggs and embryos (Zierler et al., 1985). On the other hand, D N A polymerase ~ participates in mitochondrial D N A replication (Hfibscher, 1983). Mitochondria contain only the polymerase, but the enzyme was not always found in only mitochondria: large portions of the enzymes were detected in the soluble fraction in rat liver and HeLa cells (Bolden et al., 1977) and some of the enzymes were recovered in the nucleus in chick embryos and rat brain (Bertazzoni et al., 1977; Hiibscher et al., 1977). Thus, further studies are required to elucidate the subcellular localization of D N A polymerase ~,. It is not well known whether the enzyme activity changes with mitochondrial D N A replication or not. During animal embryogenesis, it was found that a large amount of D N A polymerase c¢ accumulates in the cytoplasm of unfertilized eggs (Fansler and Loeb, 1969; Margulies and Chargaff, 1973; Shioda et al., 1977; Nagano et al., 1982). The enzyme migrates to the nucleus to participate in nuclear, D N A replication, which oocurs at an extremely rapid rate after fertilization (Fansler and Loeb, 1969; Loeb and Fansler, 1970; Shioda et al., 1980; 'Shioda and Nagano, 1983). As for D N A polymerase y, Fox et fd.
(1980) have reported that almost all the enzyme is localized in mitochondria in Xenopus eggs, and that the level of the activity per embryo remains constant throughout early embryogenesis. Similar results were obtained for teleost fish embryos (Mikhailov and Gulyamov, 1983). On the contrary, in sea urchin embryos, Gadaleta et al. (1977) have reported that mitochondrial D N A polymerase activity remains constant until the early blastula stage and then it rises, reaching a maximum at the time of hatching; thereafter it decreases to 50% of the maximum level, remaining constant up to the gastrula stage. Habara et al. (1979) found D N A polymerase 7 in the sea urchin nuclear fraction, although subcellular localization of the enzyme was not characterized. Thus, different viewpoints on behaviour of D N A polymerase ~: during embryogenesis were drawn in frog and sea urchin embryos. In the present study, we examined subcellular localization of D N A polymerase ~, and changes in its activity during embryogenesis in relation to mitochondrial D N A replication, in sea urchin eggs and embryos. The results revealed that the enzyme is localized predominantly in mitochondria and that the enzyme activity per embryo increases during embryogenesis, whereas the activity per mitochondrial D N A is kept constant. Difference in behaviour of the enzyme between frog and sea urchin embryos is also discussed. MATERIALS AND METHODS
Eggs and embryos Gametes of the sea urchin, Hemicentrotus pulcherrimus, were obtained by injection of 0.5 M KCI into the body cavity. Fertilized eggs (2 × 104ernbryos/ml) were allowed to develop at 20°C for the indicated time with gentle stirring
525
526
MASAKI SHIODA
in artificial sea-water (Jamarin Laboratory, Osaka, Japan). After washing three times each with artificial sea-water and 1.0 M glucose, the eggs or embryos were used immediately or stored at -80°C, the results being identical.
Chemicals Unlabelled deoxyribonucleoside 5'-triphosphates (dATP, dCTP, dGTP and dTTP) and [3H]dTTP (96.8 Ci/mM) were purchased from Boehringer-Mannheim (Mannheim, FRG) and New England Nuclear (Boston, Massachusetts, USA), respectively. Calf thymus DNA was obtained from Sigma (St Louis, Missouri, USA) and activated according to the method of Fansler and Loeb (1974). N-Ethymaleimide and poly(rA)'oligo(dT)m 0 were from Wako (Osaka, Japan) and Pharmacia (Uppsala, Sweden), respectively. Pancreatic RNase (RNase A) was obtained from BoehringerMannheim, and treated at 100°C for~ 15 min before use, Cell fractionation by differential or isopycnic centrifugation For cell fractionation by differential centrifugation, eggs (1.0 × 105 per ml) were homogenized in 1.0ml of a 1.2M sucrose solution containing 5 mM 2-mercaptoethanol and 5 mM MgC12. The homogenate was centrifuged at 1000g for 5 min. The supernatant was again centrifuged at 10,000 g for 30min, followed by re-centrifugation of the 10,000g supernatant at 100,000g for 90min. For each centrifugation, 2.0 M sucrose was layered on the bottom of the centrifuge tube as a cushion. Aliquots (2-10 # 1) of the supernatants or precipitates suspended in 200gl of a 0.25M sucrose solution containing 5 mM 2-mercaptoethanol and 5 mM MgC12 were assayed for DNA polymerase activities. For cell fractionation by iospycnic centrifugation, eggs (1.0 x l0 s per ml) were homogenized in 1.0 ml of a 0.25 M sucrose solution containing 5 mM 2-mercaptoethanol and 5 mM MgCI 2. The homogenate (0.3 ml) was applied onto a 4.7ml 1(L70% (w/v) linear sucrose gradient containing 5 mM 2-mercaptoethanol and 5 mM MgCI2, followed by isopycnical centrifugation at 55,000 rpm for 5 hr at 2°C, in a Hitachi swinging-bucket rotor, RPS 55T. After the centrifugation, fractions (0.13 ml) were collected from the bottom of the centrifuge tube, The buoyant density of each fraction was determined by measuring the refractive index. Aliquots (5/~1) of the fractions were assayed for DNA polymerase activities. Assay of DNA polymerases ct and [3 The activities of DNA polymerases ~t and fl were determined from the incorporation of [3H]dTMP into the acid-insoluble materials. The standard reaction mixture contained 50 mM Tris-maleate buffer (pH 8.0), 7 mM MgC12, 20/~M each of dATP, dCTP and dGTP, 10/~M dTTP containing 0.4/zCi [3H]dTTP (400 cpm = 1 pmol dTTP), 40 mM NaCI, 2.5/~g activated DNA, 10#g bovine serum albumin and 10% glycerol in a final volume of 25/zl. Incubation was carried out in the absence and presence of 20 mM N-ethylmaleimide for 30 min at 26°C, and DNA polymerase ~t and fl activities were determined as the drug-sensitive and drugresistant ones, respectively, as described previously (Usuki and Shioda, 1986). Assay of DNA polymerase 7 Before the assays, the enzyme solutions were treated ~vith 50 mM Tris-HC1 (pH 8.0), 1.0% Triton'X-100, 250 mM NaCI, l mM spermidine, 1 mg/ml bovine Serum albumin and 5 mM EDTA (final concentrations) at 0°C for 10 mih. Incubation was carried out for 30 min at 26°C in a reaction mixture (25#1) containing 50mM Tris-maleate buffer (pH 8.5), 5 mM MgC12, 100mM NaC1, 20 mM potassium phosphate (pH 7.9), 1.0#M dTTP containing 1.0/~Ci [3H]dTTP (10,000cpm = 1 pmol dTTP), 0.5#g poly(rA). oligo(dT)10, I0 #g bovine serum albumin and 10% glycerol (final concentrations). After the incubation, 25/~1 of a carrier solution containing 200mM Tris-HC1 (pH 8.0),
1.0% sodium dodecyl sulphate, 0.5M NaCI, 400mM EDTA, 100#M each of dATP, dCTP, dGTP and dTTP, and 4 mM ATP was added to stop the reaction and to reduce nonspecific binding of the radioactivity to the filter paper. The radioactivity incorporated into the acidinsoluble materials was determined as described previously (Shi0da et al., 1980). To assure the ?-polymerase activity, incubations were carried out in the absence and presence of 20 mM N-ethylmaleimide, and DNA polymerase ? activity was determined as the drug-sensitive activity.
Measurement of DNA, RNA and protein contents In each fraction prepared by isopycnic centrifugation as described above, nucleic acids were extracted as described previously (Dillon et al., 1985), and RNA content was determined by the method of Mejbaum (1939). The fractions of nucleic acids in 50mM Tris-HCl (pH 7.5) and 5mM MgCI 2 were treated with pancreatic RNase (0.05 mg/ml) at 37°C for 60 min, followed by NaOH treatment (0.3N) at 37°C for 17hr. After neutralization with 1.0 N HC1, DNA was extracted as described (Dillon et al., 1985), and the content of DNA was determined by the method of Burton (1956). To determine the content of mitochondrial DNA, the fractions prepared by the isopycnic centrifugation were treated with EDTA (5 mM), stood for 10 hr at 4°C and then centrifuged at 10,000g for 30min. The precipitate was dissolved in 50 #1 of 1.0% sodium dodecyl sulphate, 50 mM Tris-acetate (pH 8.0), 2 mM EDTA and 15 mM NaC1, and then subjected to 0.7% neutral agarose gel electrophoresis as described previously (Shioda, 1986a). The amount of mitochondrial DNA in the gel was determined by comparison with that of standard DNA, using a densitometer. As the standard DNA, mitochondrial DNA was isolated from unfertilized eggs according to the method of Kaneko and Terayama (1975). Protein content was determined by the method of Lowry et al. (1951). RESULTS
Subcellular distribution of D N A polymerase 7 analyzed by differential centrifugation To examine the subcellular distribution of D N A polymerase 7 in the eggs, the egg h o m o g e n a t e was fractionated by differential centrifugation. In this experiment, D N A polymerase a activity was also examined as a control, since subcellular distribution of the enzyme has been characterized in sea urchin eggs (Shioda et al., 1980). Non-buffered m e d i u m was used to avoid leakage of enzymes from the organelle in which the enzymes were originally localized, as reported for D N A polymerase a (Lynch et al., 1975), a l t h o u g h the separation between different kinds of organelle became poor. W h e n a non-buffered 0.25 M sucrose solution was used, considerable a m o u n t s of D N A polymerase ~ activity were recovered in the 1000 g a n d 10,000 g precipitates, a n d the yield of the activity was poor. These results differ from our previous one, i.e. almost all the D N A polymerase was f o u n d to be localized on the r o u g h endoplasmic reticulum (Shioda et al., 1980), suggesting that cosedimentation of organella occurred t h r o u g h nonspecific binding between organella u n d e r the condition used. W h e n a non-buffered 1.2 M sucrose solution was used to prevent yolk sedimentation, 7 7 % of the D N A polymerase a was recovered in the 100,000 g precipitate (Table 1), the results thus being similar to the previous ones. This suggests t h a t
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D N A p o l y m e r a s e ), in sea urchin e m b r y o s Table 1. Subcellular distribution of DNA polymerase 7 on fractionation by differential centrifugation
Fraction 1,000 g 10,000 g 100,000 g 100,000 g
~n
D N A polymerase activity pmol (%) ct ~,
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subcellular fractionation is available to determine the localization of DNA polymerase y under these conditions. Under these conditions, 80% of the DNA polymerase ~ activity was recovered in the 10,000g precipitate (Table 1), indicating that DNA polymerase y is localized predominantly in mitochondria.
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Unfertilized eggs were homogenized in 40% sucrose, 5 mM 2-mercaptoethanol and MgCI 2, and then fractionated by differential centrifugation, followed by assaying of DNA polymerases = and ),, as described in the text.
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Fig. 1. Subcellular d i s t r i b u t i o n o f D N A polymerases, as
analyzed by isopycnic centrifugation. Unfertilized eggs were homogenized in a non-buffered solution, followed by isopycnic centrifugation, and then assaying of DNA polymerases in each fraction, as described under Materials and Methods. (©) DNA polymerase ct activity; (A) DNA polymerase fl activity; (0) DNA polymerase ~, activity; (---), absorbance at 280 nm; (x) buoyant density.
On isopycnic centrifugation with a non-buffered solution, two major peaks (a and b in Fig. 1) of 2.5 mg in peak (b). Based on this value, the purity of absorbance at 280 nm were obtained. The activity mitochondria in peak (b) was estimated to be 78%, profiles of DNA polymerases ct and ~ coincided with assuming that the ratio, protein content (mg) to DNA peak (a). Peak (a) seemed to correspond to the rough content (~g), of pure mitochondria is 1.96, as reendoplasmic reticulum fraction, judging from its ported for Xenopus egg mitochondria (Dawid, 1966). buoyant density (mean _ SD: 1.262 __+0.020 g/cm 3, These results indicate that peak (b) is the main N = 8) and content of rRNA (0.13 mg/mg protein), mitochondrial fraction. Thus, DNA polymerase ~ in as characterized previously (Shioda et al., 1980). On peak (b) is indicated to be localized in mitochondria, the other hand, distribution of DNA polymerase 7 since the buoyant densities of the enzyme and mitoactivity coincided with peak (b). The buoyant density chondria were same. DNA polymerase 7 activities per of the peak was 1.216___0.012 (mean +__SD) g/cm 3 protein content were different from each other in (N = 8), in agreement with that of mitochondria in peak (a), peak (b), fraction (c) and fraction (d), while rat liver (Beaufay et al., 1959). These results suggest the enzyme activity per mitochondrial DNA of peak that DNA polymerase y is localized in mitochondria. (b) was similar to that of peak (a) and fraction (c), but To quantify the localization of DNA polymerase differed from that of fraction (d). This suggests that in mitochondria, fractions prepared by the isopycnic DNA polymerase ~ is localized in the same kind of centrifugation in Fig. 1 were grouped tentatively into organelle (mitochondria) in peak (a), peak (b) and five fractions: bottom (precipitate), peak (a) (fraction fraction (c), but not in fraction (d). Peak (a), peak (b) number 1-9), peak (b) (fraction number 10-20), (c) and fraction (c) contained 14%, 72% and 8% of (fraction number 21-29) and (d) (fraction number DNA polymerase 7 activity. Thus, it is suggested that 30-40), and DNA and protein contents of each at least 96% of DNA polymerase ~, is localized in fraction were determined (Table 2). Peak (b) con- mitochondria. tained 73% of mitochondrial DNA and little nuclear On the isopycnic centrifugation of the homogenate DNA, whereas the bottom fraction:contained 83% of o f 15hr old embryos (late blastulae), 9%, 15%, nuclear DNA and little mitochondrial DNA. The 74% and 7% of DNA polymerase 7 activity were protein content per /zg mitochondrial DNA was recovered in the nuclear fraction (buoyant density Table 2. Characterization of each fraction prepared by isopycnic centrifugation DNA content
Fraction Bottom Peak (a) Peak (b) (c) (d)
DNA polymerase 7-activity pmol (%) 0 2.5 13.0 1.4 1.1
(0) (14) (72) (8) (6)
nuclear DNA pg (%) 1.0 (83) 0.2 (17) 0 (0) 0 (0) 0 (0)
mit DNA #g (%) 0 0.6 2.4 0.3 <0.1
(0) (18) (73) (9) (0)
Protein per mitDNA mg/pg
Protein content (mg) . 3.6 6.0 5.4 15.0
.
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),-activity per protein pmol/mg
y-activity per mitDNA pmol/pg
. 0.7 2.2 0.3 0.07
4.2 5.4 4.7 >11
Unfertilized eggs were fractionated by the isopycnic centrifugation as described in Fig. 1. Fractions corresponding to those in Fig. 1 were grouped to five fractions: bottom (corresponding to precipitate), peak (a) (fraction number 4-9), peak (b) (fraction number 10-20), (c) (fraction number 21-30) and (d) (fraction number 31-40). The grouped fractions were characterized. Nuclear DNA content was determined by subtracting the content of mitochondrial DNA from total DNA content determined by the method of Burton (1956). Each value corresponds to that from 1.0 x 106 eggs. mitDNA represents mitochnndrial DNA.
528
M A S A K I SI'IIODA
1.33 g/cm 3) (Shioda et al., 1982), rough endoplasmic reticulum fraction, mitochondrial fraction and other fraction (buoyant density less than 1.18g/cm3), respectively.
0.6
of the buffered solution. These results suggest that almost all the DNA polymerase y leaked from the mitochondria into the soluble fraction in the presence of Tris buffer. Figure 2B shows the relationship between the extent of leakage of DNA polymerase y from mitochondria and the concentration of Tris-HC1 buffer (pH 8.0); it shows that half the enzyme leaked out in the presence of less than 5 mM Tris buffer and that the leakage occurred in a concentration-dependent manner. Almost all of the enzyme also leaked out in the presence of 0.1 M NaCI or KCI without Tris buffer. On the other hand, almost all the DNA polymerase ~t and about 30% of the DNA polymerase fl remained on the endoplasmic reticulum in the presence of 20 mM Tris-HC1 buffer (Fig. 2A). Almost all of both polymerases were free from organella in the presence of 0.1 M NaCl or KCl without Tris buffer.
0.4
Changes in DNA polymerase y activity and the amount of mitochondrial DNA during embryogenesis
Leakage of DNA polymerase Y from mitochondria When the egg homogenate was prepared in the presence of a buffered solution (20 mM Tris-HCl, pH 8.0), almost all the DNA polymerase ? activity was recovered in fractions with densities lower than that of mitochondria, and the activity continued to sediment (Fig. 2A). The peak (b) material equilibrated at a Similar density to that in the absence o
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Mitochondrial fractions were prepared from 0 hr old (unfertilized eggs), 5 hr old (morulae), l0 hr old (early blastulae), 15 hr old (late blastulae), 20 hr old (gastrulae) and 25 hr old (late gastrulae) embryos by isopycnic centrifugation under the non-buffered conditions, and then the DNA polymerase Yactivity and the amount of mitochondrial DNA in each fraction determined (Fig. 3). DNA polymerase V activity per embryo remained almost constant during the first
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Fig. 2. Effect of Tris buffer on the leakage of DNA polymerase 7 from mitochondria. (A) Subcellular distribution of DNA polymerase 7 in the presence of Tris buffer on isopycnic centrifugation. Unfertilized eggs were homogenized in 0.25 M sucrose, 20 mM Tris-HCl (pH 8.0), 5 mM 2-mercaptoethanol and 5 mM MgCI2, followed by isopycnical centrifugation, as described under Materials and Methods, except that the sucrose gradient contained 20 mM Tris-HCl (pH 8.0), and then assaying of DNA polymerases. (O) DNA polymerase ~t activity; (A) DNA polymerase fl activity; (O) DNA polymerase Vactivity; ( - - - ) absorbance at 280 nm; (x) buoyant density. (B) Relationship between the Tris buffer concentration and the leakage of DNA polymerase )' from mitochondria. The mitochondrial fraction was prepared from unfertilizedeggs by isopycniccentrifugation, as described under Materials and Methods. The fraction suspended in 200pl of 0.25 M sucrose, 5 mM 2-mercaptoethanol and 2 mM EDTA was treated with the indicated concentrations of Tris-HC1 (pH 8.0), and then applied on 0.7 ml of a 1.2 M sucrose solution containing 5 mM 2-mercaptoethanol with a 2.0 M sucrose cushion, followed by centrifugation at 10,000g for 30 rain. The precipitate on the cushion was suspended in 20-30 pl of 0.25 M sucrose and 5 mM 2-mercaptoethanol, and then aliquots of the fractions obtained were assayed for DNA polymerase )'. The figure shows the activity remaining in the mitochondria. 100% corresponds to 0.3 pmol dTMP incorporated.
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(h)
Fig. 3. Changes in DNA polymerase y activity (A), the amount of mitochondrial DNA (B) and the specific activity of DNA polymerase 7 (C) during embryogenesis. Embryos were developed at 20°C. At 10 and 20 hr after fertilization, hatching and gastrulation occurred, respectively. From embryos at the indicated stages, mitochondrial fractions were prepared by isopycnic centrifugation, and then DNA polymerase 7 activity and the amount of mitochondrial DNA were determined, as described under Materials and Methods. The specific activity of DNA polymerase 7 is expressed as the ratio of the ),-activity (nmol dTMP incorporated) to the amount of mitochondrial DNA (mg). Each point and bar represents the mean + SD for three separate experiments.
DNA polymerase ~, in sea urchin embryos 10 hr after fertilization. But, thereafter, it began to increase gradually, reaching about 3-fold the level in eggs by 25 hr after fertilization (Fig. 3A). On the other hand, the amount of mitochondrial DNA per embryo was found to be increased 5 to 10 hr after fertilization, and thereafter continued to increase. By 25 hr after fertilization, it had increased to about 3-fold the level in eggs (Fig. 3B). The specific activity of DNA polymerase y (DNA polymerase activity per mitochondrial DNA) showed a slight decrease at about 10hr after fertilization, but, thereafter, returned to a similar level as that in eggs (Fig. 3C). DISCUSSION Although the subcellular distribution of DNA polymerases changed with the conditions used, the bulk of DNA polymerase 7 was recovered in the mitochondrial fraction on the differential and isopycnic centrifugation under a certain condition by which DNA polymerase ~ was distributed in the rough endoplasmic reticulum (Tables 1 and 2, Fig. 1). These results exclude the possibility of a non-specific distribution of DNA polymerase 7 caused by the low ionic conditions. Therefore, we conclude that DNA polymerase 7 is localized predominantly in mitochondria in sea urchin eggs and embryos. A similar conclusion was drawn for Xenopus eggs (Fox et al., 1980) and some mammalian cells (Tanaka and Koike, 1978; Bazzicalupo, 1979). Some studiesr suggested that some portion of DNA polymerdse ~, is localized in the nucleus (Bertazzoni et al., '1977; Hiibschcr et al., 1977; Kalf et al., 1980). In the I~resent study, a small amount of the enzyme activity was also detected in the nuclear fraction of embryos, but it Was uncertain whither the activity originated from nuclei or from mitochondria. To elucidate this localization, further studies are required. Lynch et al. (1975) showed that DNA polymerase leaks easily from nuclei with Tris buffer or KC1. Similarly, the present study indicated that DNA polymerase ), leaks from mitochondria in the presence of Tris buffer (Fig. 2). Although it is not known whether or not the leakage of DNA polymerase ), occurs to the same extent in other cells, the leakage may explain the low recovery of DNA polymerase y in the mitochondrial fraction and localization of the enzyme in the soluble fraction, reported for some cells (Bolden et al., 1977). Many contradictory results have been reported as to the proliferation of mitochondria during sea urchin embryogenesis: some indicated that mitochondrial proliferation does not occur in unfertilized eggs or embryos up to the pluteus stage (Matsumoto et al., 1974; Bresch, 1978; Rinaldi et al., 1979), but others showed that it occurs after fertilization (Shaver, 1956; Anderson, 1969; Bresch, 1973; Kaneko and Terayama, 1975). In the present study, although the exact time the increase started was uncertain, the mitochondrial DNA content per embryo was found to be increased 5 to 10 hr after fertilization (Fig. 3B). DNA polymerase 7 activity also began to increase 15 hr after fertilization (Fig. 3A). Thus, the specific activity of DNA polymerase y remained almost constant throughout embryogenesis, with a slight decrease
529
at the early blastula stage (Fig. 3C). This may imply the following: pre-existing DNA polymerase ), participates in mitochondrial DNA replication at the early blastula stage; after the replication, the doubling of mitochondria including DNA polymerase ~ occurs; thereafter, mitochondria begin to proliferate asynchronously. An essentially similar relationship has been reported for mitochondriai DNA replication and a mitochondrial enzyme, succinateeytochrome-c reductase, in embryos of this sea urchin species (Kaneko and Terayama, 1975). In Xenopus (Fox et aL, 1980) and teleost fish (Mikhailov and Gulyamov, 1983), DNA polymerase ), activity per embryo was shown to remain constant during early embryogenesis, although it increased in the present study. In the former species of embryos, the amount of mitochondria also remains constant during the stage, but it begins to increase at the later stage. The difference in the amount of the enzyme activity between the former species of embryos and the present embryos may be due to the timing of mitochondrial proliferation. Thus, the present results are comparable, rather than contradictory, to those reported for Xenopus and teleost fish embryos as to the enzyme activity per mitochondrion. Gadeleta et al. (1977) have reported on the mitochondrial DNA polymerase in sea urchin embryos. However, under the conditions they used, a considerable amount of DNA polymerase 7 is supposed to have leaked, judging from the present results (Table 2), since mitochondria were prepared in the presence of 100 mM Tris-HCl with 240 mM KC1 in their study. So, the DNA polymerase activity they reported does not seem to represent all of DNA polymerase ~ in mitochondria in vivo, although their results may reflect some physiological aspects of mitochondrial DNA replication. The specific activity of DNA polymerase remained almost constant regardless of whether mitochondrial proliferation occurred or not during embryogenesis (Fig. 3C). Similar results have been obtained for teleost fish (Mikhailov and Gulyamov, 1983). The enzyme was found in sea urchin sperm, in which mitochondrial DNA replication does not occur (Habara et al., 1980). These results indicate that mitochondrial DNA replication is not regulated by the level of DNA polymerase ), activity, although the polymerase is essential for mitochondrial DNA replication. On the other hand, the specific activity of DNA polymerase ~t changes with nuclear DNA replication during the cell cycle and cell proliferation (Usuki and Shioda, 1986). Therefore, it is suggested that different regulatory mechanisms are involved in mitochondrial and nuclear DNA replication. Acknowledgements--This work was supported by Grantsin-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan. REFERENCES
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