Mitochondrial DNA polymerase from wheat embryos

Plant Science Letters, 21 (1981) 181--192

181

© Elsevier/North-Holland Scientific Publishers Ltd.

MITOCHONDRIAL DNA POLYMERASE FROM WHEAT EMBRYOS

L. CHRISTOPHE, L. T A R R A G O - L I T V A K , M. C A S T R O V I E J O and S. L I T V A K Institut de Biochimie Cellulaire et Neurochimie, C.N.R.S. 1, rue Camille Saint SaChs,

33000 Bordeaux (France)

(Received September 17th, 1980) (Revision received December 4th, 1980) (Accepted December 4th, 1980)

SUMMARY A DNA polymerase activity isolated from purified wheat embryo mitoch0ndria has been characterized. The enzyme is confined to the matrix of the organelle as shown after treatment with digitonin and sonication. After DEAE-cellulose, phosphocellulose, DNA-cellulose and Sephacryl 200 SF chromatography, only one peak of activity has been found. The mitochondrial DNA polymerase recognizes very efficiently activated DNA as well as poly(dA-dT ~2 ), but very poorly poly(A-dT,: ) in the presence of manganese and not at all in the presence of magnesium. The wheat mitochondrial polymerase is completely resistant to N-ethylmaleimide, slightly inhibited by ddTTP and ethidium bromide. The template specificity and the effect of inhibitors on this polymerase are very different to those described for the animal mitochondrial DNA polymerase. We conclude that the plant mitochondrial DNA polymerase is not a DNA polymerase of the 7 type.

INTRODUCTION In 1963 it was demonstrated that the presence of fibres of the size order of DNA in the matrix of mitochondria of embryonic cells. These fibres were degraded by DNAase suggesting that they were DNA [ 1]. It is well established now that mitochondrial DNA is generally a double stranded covalently closed circle varying in length from about 5 ~m for most animal species to 30 g m or so in higher plants. Mitochondria possess the complete molecular machinery for the replication and transcription of its DNA as well as for protein synthesis. The DNA replicating and transcribing systems in mitochondria and the machinery of protein synthesis are basically similar to those in the nucleus and cytoplasm, but distinct and different in detail [2,3]. Very recently it has been found that mitochondrial genetic code is somehow different to the one directed by the nuclear genome [4--6].

182 Mitochondrial DNA is replicated by a DNA polymerase DNA-dependent which has been well characterized in mammalian cells [ 7--9]. The animal enzyme or DNA polymerase 7 is confined to the soluble mitochondrial matrix, although its presence in purified nuclei has been reported. It is n o t clear at the present time if its presence in the nuclear fraction has something to do with the replication of the nuclear chromosome. Animal mitochondrial DNA polymerase is clearly different from the polymerase responsible for nuclear DNA replication (DNA polymerase a) following the differential inhibition of both enzymes by several reagents like ddTTP, ethidium bromide and aphidicolin [10]. Much less information is available a b o u t the plant mitochondrial DNA polymerase. While we were characterizing the multiple DNA polymerases from quiescent wheat embryos, we made a preliminary study of a DNA polymerase isolated from purified mitochondria [ 11]. This is, to our knowledge, the only report in the literature a b o u t a plant mitochondrial DNA polymerase. In this article we describe in more detail the purification and characterization of a DNA polymerase DNA-dependent from purified wheat e m b r y o mitochondria. The properties of this enzyme are compared to the animal mitochondrial DNA polymerase. MATERIALS Wheat seeds (variety Cesar) were obtained from the Brosse Monceaux Agronomical Center, Montceaux, France. Commercial wheat germ was a kind gift of the Grands Moulins de Bordeaux. Labelled products were purchased from New England Nuclear, Europe. Unlabelled nucleoside triphosphates were from Sigma Chemical Co. Polynucleotides and calf t h y m u s DNA were from Sigma, Boehringer-France or P.L. Biochemicals. Ethidium bromide and N-ethylmaleimide were from Sigma. ddTTP was from Boehringer-France. Aphidicolin was a kind gift of Dr. A.H. Todd, Imperial Chemical Industries Limited, U.K. Blue dextran was from Pharmacia. METHODS

Wheat embryo isolation E m b r y o s were separated from the rest of the grain by the m e t h o d of Johnston and Stern [12]. They were stored for several months at 4°C in the presence of anhydrous CaC12. They were more than 95% viable under the above conditions. Isolation o f mitochondria We have used essentially the m e t h o d of Cunningham and Gray [13]. Wheat e m b r y o s or fresh commercial wheat germs were homogenized with 5 vol. of buffer I (0.44 M sucrose, 50 mM Tris--HCl {pH 7.9), 3 mM EDTA,

183 1 mM 2-mercaptoethanol and 0.1% bovine serum albumin) in an ice-cold mortar for 10 rain. The crude homogenate was filtered through four layers of cheesecloth and centrifuged at 1000 × g for 6 min. The pellet was discarded and the supernatant was centrifuged twice at 2000 × g for 10 min. The supernatant was centrifuged at 18 000 X g for 20 rain. The pellet was resuspended in buffer I in a Potter-Elvejhem homogenizer (Teflon-glass) and centrifuged at 1000 × g for 6 min and then at 2000 X g for 6 min. The supernatant was pelleted at 18 000 × g for 20 min. This crude mitochondrial pellet was resuspended in buffer I and layered o n t o a discontinuous sucrose gradient (5 ml 2.2 M sucrose, 12 ml 1.45 M sucrose, 12 ml. 1.15 M sucrose and 4 ml sample. All sucrose solutions were done in buffer I). Centrifugation was performed in the vertical rotor SV-288 of the RC5 Superspeed Sorval centrifuge at 20 000 rev./min for 15 min. All operations were carried o u t at 4°C. The mitochondrial layer was recovered with a Pasteur pipette from the border between the 1.45 and 1.15 M Layers. This fraction was diluted slowly under gentle shaking with 2 vol. of buffer II (buffer I minus bovine serum albumin and 2-mercaptoethanol) and pelleted at 18 000 × g for 20 min. I

Electron microscopy The wheat mitochondrial pellet was processed as described previously for animal mitochondria [ 14].

Preparation of templates Calf t h y m u s DNA was activated by mild digestion with DNAase I [15]. Synthetic polynucleotide solutions were 2.4 A260 units/ml in 10 mM Tris-HC1 (pH 7.5). After mixing the appropriate polynucleotides, the solution was heated at 75°C for 15 min (except for poly(dC-dG~2) or poly(C-dG12) which were heated at 90°C) and left at room temperature for 30 min. The template primer ratio was 5 : 1.

Succinate dehydrogenase assay The m e t h o d of Singer et al. was used [16].

Fumarase assay The m e t h o d of Massey was used (17).

Purification of wheat mitochondrial DNA polymerase (a) Enzyme solubilization. Mitochondria obtained as described above and suspended in buffer II at a concentration of I mg/ml of protein, was treated with digitonin at a final concentration of 0.5 rag digitonin per mg of protein and incubated for 5 min at 0°C. Digitonin treatment was stopped b y dilution with 5 vol. of buffer II. Then, after the Triton X.100 (0.5%) was added, the suspension is sonicated in an Annemase disintegrator at m a x i m u m intensity, thrice for 10 s with intervals of I rain. The sonicated

184 solution is centrifuged at 18 000 X g for 20 min and the supernatant submitted to different chromatographic steps. (b) Chromatographic steps. (1) DEAE-cellulose. The column (1.6 × 6 cm) is equilibrated in buffer III (50 mM Tris--HC1 (pH 7.9), 1 mM 2-mercaptoethanol, 0.1 mM EDTA, 20% glycerol and 0.2% Nonidet P-40). After the sonicated extract has been absorbed and the column washed extensively, the enzyme is eluted with 0.3 M KC1 in buffer III. (2) Phosphocellulose. The column of phosphocellulose (1.5 X 6 cm) is equilibrated in buffer III. DNA polymerase is eluted by a KC1 gradient between 0 M and 0.8 M. (3) DNA cellulose. DNA cellulose was prepared by the m e t h o d of Alberts and Herrick [28]. The column (0.5 X 2.5 cm) was equilibrated with buffer IV (20 mM potassium phosphate (pH 7.5), 1 mM 2-mercaptoethanol, 0.2% Nonidet P-40 and 20% glycerol). The enzyme was eluted with a gradient between 20 mM and 600 mM potassium phosphate in buffer IV. (4) Sephacryl 200 SF. The column (2.5 X 97 cm) was equilibrated and the different proteins eluted with buffer V (150 mM potassium phosphate (pH 7.5), 1 mM 2-mercaptoethanol and 20% glycerol). The void volume was determined with Blue Dextran. (5) DNA polymerase assay. The incubation mixture contained in a final volume of 50 ~l the following components: 50 mM Tris--HC1 (pH 7.9), 50/~M dCTP, dGTP, dATP, 10 pM [3H]TTP ( 1 0 0 0 2 0 0 0 cpm/pmol), 5 pg bovine serum albumin, 4 pg activated DNA, 5 mM MgC12 and 1--5 pg of enzyme protein. The mixture is incubated for 30 min at 37°C. The reaction is stopped by addition of 1 ml of ice-cold 10% trichloroacetic acid plus 100 mM sodium pyrophosphate. Radioactive DNA is collected onto nitrocellulose membranes (Schleicher and Schuell 0.45 pm), washed with 2% cold trichloroacetic acid, dried and counted in a PPO-POPOP-toluene scintillation mixture. When synthetic polynucleotides (0.02 A260 units) were used as templates, the incubation mixture was the same except that only the corresponding precursor was present at a final concentration of 10 pM. RESULTS AND DISCUSSION

Purity of wheat mitochondria Purified mitochondria shows no contamination with other subcellular c o m p o n e n t s when observed by electron microscopy as seen in Fig. 1. We have also determined the activity of t w o specific mitochondrial enzymes, succinate dehydrogenase and fumarase. These two activities were found at a very high level in the pure mitochondrial pellet. The presence of fumarase, an enzyme of the soluble matrix indicates that the organelle structure has been preserved all throughout the purification procedure (not shown). With the use of vertical rotor we have speeded up noticeably the purification of mitochondria.

Enzyme localization We have used the glycosilated steroid digitonin to study the subcellular

185

Fig. 1. Electron microscopy of wheat mitoehondria. (x26 700). The mitoehondrial fraction is prepared as described in Methods.

location of the polymerase. This substance is able to destroy the external membrane of mitochondria, thus releasing the proteins localized between the external and the internal mitochondrial membranes [18]. As seen in Fig. 2, even at high concentrations of digitonin, the DNA polymerase activity of the organeUe is preserved, indicating that the enzyme is probably located in the mitochondrial matrix. This is the same situation found with the mammalian mitochrondrial DNA polymerase 7 [8].

Enzyme purification The enzyme was released from the organelle by sonication in the presence of detergent. These results plus the resistance to digitonin m a y indicate that the enzyme is associated to the internal mitochondrial membrane. After solubilization the enzyme was submitted to different chromatographic steps; Fig. 3 shows the behaviour of this enzyme in phosphocelltflose (Fig. 3A), DNA cellulose (Fig. 3B) and Sephacryl (Fig. 3C) columns. In all cases only

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Fig. 2. Effect of digitonin treatment on mitochondrial DNA polymerase activity. Purified mitochondria are treated with digitonin as described in Methods. After the incubation with the detergent has been stopped as in the text, the suspension was centrifuged for 15 min at 18 000 rev./min. An aliquot of the supernatant was assayed directly for DNA polymerase activity (supernatant) while the pellet was resuspended and sonicated as described in Methods prior to the polymerase assay (mitochondrial pellet). Triton X-100 was not present in the buffer used to end the incubation with digitonin and was added before the sonication step. No effect of digitonin on DNA polymerase activity per se was observed.

o n e p e a k o f a c t i v i t y was o b s e r v e d i n d i c a t i n g t h a t , as in t h e case o f t h e a n i m a l m i t o c h o n d r i a l D N A p o l y m e r a s e , o n l y o n e m a j o r a c t i v i t y is p r e s e n t in t h e organelle. T h e m o l e c u l a r w e i g h t o f t h e w h e a t m i t o c h o n d r i a l p o l y m e r a s e , d e t e r m i n e d b y gel f i l t r a t i o n , was f o u n d t o be 180 0 0 0 (Fig. 3D). I t is interesting t o n o t e t h a t w h e n t h e s u p e r n a t a n t f r o m s o n i c a t e d m i t o c h o n d r i a was f i l t e r e d t h r o u g h S e p h a c r y l , w i t h o u t a p r e v i o u s D E A E - c e l l u l o s e step, t w o p e a k s w e r e o b t a i n e d , o n e at t h e level o f 1 8 0 0 0 0 a n d t h e o t h e r c o i n c i d i n g w i t h t h e v o i d v o l u m e ( n o t s h o w n ) . T h i s last p e a k c o r r e s p o n d s m o s t p r o b a b l y t o t h e m t D N A - e n z y m e c o m p l e x , since it d i s a p p e a r s a f t e r D E A E - c e l l u l o s e c h r o m a t o g r a p h y . T a b l e I gives t h e s u m m a r y o f a t y p i c a l p u r i f i c a t i o n p r o c e d u r e . We h a v e n o t i n c l u d e d t h e p h o s p h o c e l l u l o s e a n d D N A cellulose s t e p s in this s u m m a r y b e c a u s e , a l t h o u g h t h e s e s t e p s led to t h e e l i m i n a t i o n o f c o n t a m i n a t i n g p r o t e i n s , t h e specific a c t i v i t y o f t h e e n z y m e was n o t i m p r o v e d .

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Fig. 3. Chromatographic behaviour of wheat mitochondrial DNA polymerase. (A): chromatography of the mitochondrial polymerase on a phosphocellulose column. Elution was performed with a KCI gradient. Ten ~1 of each fraction were incubated 30 rain in the presence of poly(dA-dT~ 2) and Mg2÷, as described in Methods. B: chromatography in a DNA cellulose column. Elution was performed by a potassium phosphate gradient. C: gel filtration on a Sephacryl-200 SF column. The conditions are described under Methods. D: molecular weight determination. The mitochondrial DNA polymerase was chromatographed through a Sephacryl-200 SF column as described in the text. Proteins used as standards were: 1, RNAase A (13 700); 2, chymotrypsinogen A (25 000); 3, ovoalbumin (43 000); 4, bovine serum albumin (67 000); 5, aldolase (158 000); 6, catalase (232 000).

Characterization o f wheat mitochondrial DNA polymerase T h e m i t o c h o n d r i a l D N A p o l y m e r a s e f r o m w h e a t e m b r y o s , as well as all t h e o t h e r e u k a r y o t i c D N A p o l y m e r a s e s , is n o t able t o r e c o g n i z e native D N A , while D N A a s e - a c t i v a t e d D N A is a v e r y g o o d t e m p l a t e f o r t h e e n z y m e (Table II). S o m e s y n t h e t i c p o l y n u c l e o t i d e s can also be very e f f i c i e n t t e m p l a t e s ,

188 TABLE I P U R I F I C A T I O N OF MITOCHONDRIAL DNA POLYMERASE A unit of enzyme is the amount able to catalyse the incorporation of 1 pmol of [3H]TMP with poly(dA-dT 12 ) as template in 30 rain. Procedure

Volume (ml)

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Activity (units)

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like poly(dA-dT12) or poly(dC-dG12). Poly(A-dT,2), the best template for the animal mitochondrial polymerase, is n o t copied by the wheat enzyme under our experimental conditions, when magnesium ions are present; in the presence of manganese some activity was detected. No activity was f o u n d with poly(C-dG,2 ), neither in the presence of magnesium nor manganese. In general, it can be seen that the enzyme activity is better in the presence of Mg 2+ than Mn 2+. The optimal pH and temperature of the mitochondrial DNA polymerase from wheat e m b r y o s are shown in Fig. 4; best activity with poly(dA-dT12 ) was found around pH 8 and 35°C. These values are quite similar to those obtained with the animal mitochondrial polymerase.

TABLE II TEMPLATE SPECIFICITY OF MITOCHONDRIAL DNA POLYMERASE Results are given in pmol incorporated during 30 min. About 5 ~g of mitochondrial e n z y m e p r o t e i n w e r e used.

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The use of specific inhibitors has been extremely useful in characterizing the different DNA polymerases found in eukaryotic cells. We have done a detailed study of the inhibition of animal and plant DNA polymerases using four inhibitors: (a) N-ethylmaleimide, that blocks the --$H groups of cysteine, inhibits enzymes which have essential cysteine groups in their active sites; (b) ddTTP, a precursor analog which has proven to be a very powerful inhibitor of the animal mitochondrial enzyme, while it does not affect the activity of DNA polymerase a which replicates the nuclear chromosome [19]; (c) ethidium bromide, a DNA intercalating drug, which inhibits strongly the animal mitochondrial DNA polymerase under conditions where the other polymerases are much less affected [20,21] and (d) aphidicolin, a drug that inhibits specifically DNA polymerase a [22]. As seen in Fig. 5, the behaviour of plant and animal mitochondrial DNA polymerases is quite different and only in the case of aphidicolin the situation with both enzymes is similar. The plant mitochondrial DNA polymerase is very resistant to N-ethylmaleimide and to ethidium bromide, as seen in Figs. 5A and 5C. The same inhibition was observed when poly{A-dTt2) was used as template in the presence of Mn 2+. These last results are very different to those obtained with the other DNA polymerases purified from wheat embryos which are strongly stimulated by ethidium bromide when poly(A-dT~2) is used as template and the incubation performed at 37°C [23]. The TTP analog, ddTTP, inhibits the plant enzyme, but at a lower extent than its animal counterpart (Fig. 5B). Aphidicolin which inhibits strongly animal D N A polymerase a and a recently described a-like D N A polymerase from plant cells,does not affect the activities of the animal and plant mitochondial polymerases (Fig. 5D).

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CONCLUSION

Both prokaryotic and eukaryotic organisms have multiple D N A polymerases. In the case of plant D N A polymerases we have described some important differences with the animal D N A polymerases a, ~ and 7 [ 11 ]. We have isolated, purifed and characterized one major form of D N A poly-

191

merase activity from wheat mitochondria. Although the organeUe structural features are somehow better in mitochondria obtained from wheat embryos than from commercial wheat germ, as observed by electron microscopy, the DNA polymerase activity from both sources is identical following all the criteria used in this article. Thus, if wheat embryo is the best source when a high yield of intact mitochondria is needed, commercial wheat germ, because of its abundance, is a very good source for the purification of important amounts of enzyme. The presence of minor species of DNA polymerases in wheat mitochondria cannot be excluded, but all our results point to the presence of a unique DNA polymerase in plant mitochondria. This enzyme catalyses the same reaction as its animal counterpart, but both enzymes have very different properties following several criteria. For instance, the template specificity and the inhibition by N-ethylmaleimide, ethidium bromide and ddTTP are very different for the animal DNA polymerase ~/and the wheat mitochondial DNA polymerase. We conclude, thus, that the plant mitochondrial polymerase is not a ~/-like DNA polymerase. However, this last type of enzyme exists in plant cells as we reported some time ago [ 24]. The ~/-like DNA polymerase we described in wheat embryos may correspond to the chloroplastic DNA polymerase, since it has been claimed very recently that the DNA polymerase purified from spinach chloroplasts shares some properties with animal DNA polymerase ~/ [ 25]. It is not surprising that these differences are observed between the animal and plant mitochondrial enzymes, since both types of organelles may have some striking structural and functional dissimilarities. For instance, DNA from plant mitochondria can be up to 7 times bigger than the one from animal organelles. Some properties of the wheat mitochondrial enzyme, like template specificity and chromatographic behaviour, are similar to those of wheat DNA polymerase B [ 11], but the size and inhibition by agents like N-ethylmaleimide and ethidium bromide are different from both enzymes. In the case of animal cells some minor differences have also been observed between the mitochondrial and nuclear DNA polymerase ~/ [7]. Assuming that both proteins have a common ancestor the differences may be explained by the specific maturation needed for the import of mitochondrial proteins [26]. A very important question which is being asked by many groups interested in the problem of mitochondiral biogenesis, concerns the interaction between the nuclear and the mitochondrial genetic systems. If we assume that plant mitochondrial DNA polymerase is coded in the nuclear genome, as in the case of yeast 'petite' mutants which have DNA incapable of coding for any mitochondrial protein, but still have normal amounts of mitochondrial DNA polymerase [27], we can ask the question of how the cytoplasmically synthetized mitochondrial enzyme is transported into the organelle and functionally integrated there. The fact that the plant mitochondrial DNA polymerase can be differentiated from the other plant cell polymerases following several criteria and the nice property of the wheat embryo system

192 o f b e i n g n a t u r a l l y s y n c h r o n i z e d a f t e r w a t e r i m b i b i t i o n , has p r o m p t e d us t o c h o o s e t h e w h e a t e m b r y o as a m o d e l f o r t h e s t u d y o f t h e o r i g i n o f t h e m i t o c h o n d r i a l r e p l i c a t i n g m a c h i n e r y . R e s u l t s o n t h a t sense are u n d e r p r o g r e s s . ACKNOWLEDGEMENT T h e a u t h o r s a r e g r a t e f u l t o Drs. B. G u e r i n a n d J. V e l o u r s f o r a d v i c e in t h e p r e p a r a t i o n o f m i t o c h o n d r i a . T h i s w o r k was s u p p o r t e d in p a r t b y t h e Univ e r s i t y o f B o r d e a u x II a n d C . N . R . S . REFERENCES 1 2 3 4 5

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

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