Replicative Deoxyribonucleic Acid Synthesis in Isolated Mitochondria ...

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Feb 21, 1977 - mitochondria isolated from grande and petite (rho-) strains were .... In experiments involving petite strains, cells ..... The DNA synthetic ac-.

Vol. 130, No. 3 Printed in U.S.A.

JOURNAL OF BACTRIUOLOGY, June 1977, p. 973-982 Copyright C 1977 American Society for Microbiology

Replicative Deoxyribonucleic Acid Synthesis in Isolated Mitochondria from Saccharomyces cerevisiae JOHN S. MATTICK AND RUTH M. HALL* Department of Biochemistry, Monash University, Clayton, Victoria, 3168, Australia Received for publication 21 February 1977

The characteristics of a system for the in vitro synthesis of mitochondrial deoxyribonucleic acid (mtDNA) in mitochondria isolated from Saccharomyces cerevisiae are described. In this system the exclusive product of the reaction is mtDNA. Under optimal conditions the initial rate of synthesis is close to the calculated in vivo rate; the rate is approximately linear for 20 min but then decreases gradually with time. DNA synthesis proceeds for at least 60 min and the de novo synthesis of an amount of mtDNA equivalent to 15% of the mtDNA initially present is achieved. The rate and extent of synthesis observed with mitochondria isolated from grande and petite (rho-) strains were similar. The mode of DNA synthesis is semiconservative; after density labeling with 5bromodeoxyuridine triphosphate, in vitro, the majority of labeled DNA fragments of duplex molecular weight, 6 x 106, are of a density close to that calculated for hybrid yeast mtDNA. The density label is incorporated into one strand of the duplex molecules. These properties indicate that the synthesis resembles replicative rather than repair synthesis. This system therefore provides a convenient method for the study of mtDNA synthesis in S. cerevisiae. The observation that mtDNA synthesis is semiconservative in vitro suggests that the dispersive mode of synthesis observed in S. cerevisiae in vivo labeling studies is the result of some other process, possibly a high recombination rate.

Mitochondrial deoxyribonucleic acid (mtDNA) exists as a separate genetic system within the eukaryotic cell and the mechanism and control of mtDNA synthesis are therefore of fundamental interest to the areas of mitochondrial biogenesis and genetics. Since the fungi, and in particular the unicellular yeast Saccharomyces cerevisiae, have provided a most convenient system for genetic and biochemical analysis of mitochondrial function, the mechanism of replication of yeast mtDNA is of particular importance. It is well established that mtDNA synthesis takes place within mitochondria, and that mtDNA replication is at least partly independent of nuclear DNA replication (see reference 4). However, very little is known about the mechanism of replication of fungal mtDNA. Whereas in vivo density-labeling studies have established that the mode of mtDNA synthesis in metazoa is semiconservative (6), density-labeling experiments in vivo reveal a dispersive rather than a semiconservative pattern in Neurospora crassa (21) and S. cerevisiae (27). Dispersive mtDNA synthesis has also been observed in Euglena gracilis (22). This finding does not preclude the possibility that mtDNA

replication is indeed semiconservative, as an apparent dispersive pattern could arise as a result of an extremely high rate of recombination of MtDNA, and indeed evidence for extensive recombination of yeast mtDNA in vivo has been reported (23). However, such effects render the study of mtDNA synthesis in vivo extremely difficult. To avoid problems encountered with in vivo studies, we have developed an in vitro system for mtDNA synthesis in isolated mitochondria from S. cerevisiae. The characteristics of this system are described in detail in this paper. In contrast to the systems for yeast mtDNA synthesis reported previously (28, 29, 30), extensive synthesis of mtDNA is observed. Furthermore, evidence that the observed mtDNA synthesis has the features of replicative rather than repair synthesis is presented. We have also shown that the mode of DNA synthesis is semiconservative. This system provides a convenient method for the study of mtDNA synthesis in S. cerevisiae, which we have already used to demonstrate a functional association of mtDNA synthesis with the mitochondrial membrane (11) and to study the effects of petite-inducing agents on mtDNA synthesis (19). 973

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lated as a relatively undamaged fraction. Cells were labeled by the addition of 14C-labeled Strains. S. cerevisiae strains used in these studies protein hydrolysate (15 MCi/liter) to the growth media. For chemostat cultures, isotope was added 2 h were L2200 (a trpl adel Iys2; rho+), L411 (a ura his; rho+), and petite strains K45 (a ura his; rho-) and before harvesting. Prelabeling in this way produced E5 (a ura his; rho°), derived from L411. All strains mitochondrial protein with a specific activity of 5,000 to 10,000 14C dpm/mg of protein for batch culhave been described previously (18). Chemicals. Deoxythymidine triphosphate tures and 2,000 to 6,000 14C dpm/mg of protein for (dTTP), deoxyadenosine triphosphate (dATP), deox- chemostat cultures. Preparation of mitochondria. Cells were conyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), and olivomycin were purchased verted to protoplasts and mitochondria were isofrom Schwarz/Mann, Orangeburg, N.Y., and adeno- lated as previously described (5). Mitochondria were sine triphosphate (ATP), phosphocreatine, creatine washed twice with 4ce-cold 0.6 M sorbitol, 10 kinase (type 1, rabbit muscle), ribonucleases (type mM tris(hydroxymethyl)aminomethane(Tris)-hydro1-A, bovine pancreas, and T,, Aspergillis oryzae), a- chloride, 1 mM ethylenediaminetetraacetic acid amylase (type 2-A, Bacillus subtilis), actinomycin (EDTA), pH 7.4, and once with 0.6 M sorbitol, 10 D, and ethidium bromide from Sigma Chemical mM Tris-hydrochloride, 35 mM MgCl2, pH 7.4. The Co., St. Louis, Mo. Deoxyribonucleases (DNase) I mitochondrial pellet was carefully suspended in a (bovine pancreas) and II (porcine spleen) were small volume of the latter buffer, to a final concenpurchased from Worthington Biochemicals Corp., tration of approximately 50 mg of protein/ml. Assay for DNA synthesis in isolated mitochonFreehold, N.J., and Pronase (B grade, Streptodria. Mitochondria were incubated at 280C in a reacmyces griseus) from Calbiochem Pty. Ltd., Sydney, N.S.W., Australia. Sarkosyl-NL30 was a gift from tion mixture containing 50 IAM each of dATP, dGTP, Geigy Chemical Corp., Melbourne, Victoria. 5- and dCTP, 25 AsM [3H]dTTP (specific activity, 500 to Bromodeoxyuridine triphosphate (dBUTP) was pur- 1,000 Ci/mol), 1.5 mM ATP, 8 mM phosphocreatine, chased from Boehringer GmBH, Mannheim. Yeast 100 jg of creatine kinase per ml, 35 mM MgCl2, 60 extract was purchased from Difco Laboratories, mM KCl, and 20 mM HEPES buffer (N-2-hydroxyDetroit, Mich., and daunomycin from May and ethyl piperazine-N'-2-ethanesulfonic acid), pH 7.4 Baker Pty. Ltd., Melboume, Victoria. Berenil was (2800). For density labeling, dTTP was replaced by kindly provided by H. Loewe, Farbwerke Hoechst, 50 AM dBUTP, and 30 ,uM [3H]dATP (specific activity, 500 to 1,000 Ci/mol) was used as the isotopic Frankfurt. [3H]dTTP, [3HldATP, [14C]adenine, and [14C]pro- label. The reaction mixture volumes generally used tein-hydrolysate were purchased from the Radio- were 0.25 and 0.5 ml. The reaction was started by the addition of concentrated mitochondrial suspenchemical Centre, Amersham, Bucks. Whatman glass fiber disks (25-mm diameter, GF/ sion (0.04 ml per ml of reaction mixture) to preC) and paper disks (25-mm diameter, 3MM) were warmed reaction mixture. The final protein concenpurchased from W & R Balston Ltd., Maidstone, tration was 1 to 3 mg/ml. DNA synthetic activity of mitochondria is not Kent. Media and cell culture. Cells were grown in a significantly reduced by storage of the concentrated 0.5% yeast extract-salts medium (5). Additional mitochondrial suspension at 20C for at least 4 h or auxotrophic growth requirements were added at the at -70'C for at least 3 weeks. Results presented in following concentrations: tryptophan, lysine, and this paper, however, were all obtained with freshly histidine (50 gg/ml); uracil and adenine (25 Mg/mi). prepared mitochondria. Bacterial contamination of Glucose was used as the carbon source, at concentra- the mitochondrial preparation was routinely found tions of 1% for respiratory-competent (grande) cells to be less than 10' bacteria per ml of reaction mixand 2% for respiratory-deficient (petite) cells. ture. Addition of Escherichia coli up to concentraRespiratory-competent cells were routinely tions of 5 x 107 cells/ml of reaction mixture did not grown aerobically in batch culture and harvested at affect the observed activity. a cell density of 3.4 mg (dry weight) per ml (late log Duplicate 0.05-ml samples were withdrawn at phase of oxidative growth). various times and placed onto 3MM paper disks, In experiments involving petite strains, cells which were immediately immersed in ice-cold 7% were grown in glucose-limited chemostat culture. trichloroacetic acid containing 1% sodium pyrophosConditions were as follows: medium was as described phate. Samples on the disks were washed five times above, the working volume was 4 liters, and the by immersion in ice-cold 5% trichloroacetic acid (20 dilution rate was 0.1/h (generation time, 7 h). Cells min each) and once in water and then were dried. were harvested not less than 48 h after a steadyPrecipitates on filters were solubilized with Nuclear state cell density had been attained. Steady-state Chicago solubilizer, and counted in a Philips liquid cell densities were approximately 5.0 and 2.4 mg scintillation analyzer. The amount of incorporated (dry weight) of cells/ml for grande and petite cells, dTTP (dATP) was calculated from the specific activrespectively. It has been shown that, under these ity, the determined counting efficiency, and 3H conditions, the concentration of glucose in the cul- counts per minute corrected for overlap from 14C ture is maintained below the level necessary to in- radioactivity. duce catabolite repression (17). Mitochondria from The protein concentration was assayed by a modipetite cells derepressed in this way exhibit a consid- fication of the biuret method (9), and the specific erable degree of internal structure and may be iso- activity of mitochondrial protein (14C dpm/mg) was MATERIALS AND METHODS

VOL. 130, 1977

DNA SYNTHESIS IN ISOLATED YEAST MITOCHONDRIA

determined. This value was used to normalize the 3H incorporation values with respect to protein concentration. Estimation of the DNA content of isolated mitochondria. Total DNA in mitochondrial preparations was determined as follows: a portion of the mitochondrial suspension was precipitated with cold 7% trichloroacetic acid, and the pellet was extracted with 70% ethanol and then ethanol-ether (3:1) and was dried. Ribonucleic acid (RNA) was removed by hydrolysis with 0.5 M NaOH at 400C for 1 h followed by precipitation with cold 5% perchloric acid. DNA was extracted from the pellet by treatment three times with 9% perchloric acid at 700C for 15 min. The extracts were pooled, and the total DNA was assayed by the Burton diphenylamine method modified according to Giles and Myers (8). The protein concentration of the mitochondrial preparation was determined as above. The amount of mitochondrial DNA per milligram of protein was derived by correcting total DNA for the percentage of mitochondrial DNA present, as determined by analytical ultracentrifugation of DNA purified from the mitochondrial preparation. Extraction of mitochondrial DNA labeled in vitro. To extract in vitro labeled mitochondrial DNA, the reaction was stopped by the addition of EDTA (final concentration 100 mM) to the reaction mixture. Sarkosyl-NL30 was then added to a final concentration of 1%, and the mixture was incubated in the presence of 1 mg of Pronase per ml (self-digested at 370C, 30 min) at 370C for 2 h. Pronase (1 mg/ml) was again added, and the solubilized mitochondrial suspension then was dialyzed overnight at 40C against two changes of 10 mM Tris-hydrochloride, 1 mM EDTA, pH 8.0. Extracted DNA was used without further purification for preparative CsCl gradient analysis. Virtually, quantitative recovery of acid-precipitable radioactivity was achieved. In experiments with bromouracil-labeled DNA, DNA was mechanically sheared by forcing the DNA solution under high pressure through a 27-gauge syringe needle. This procedure was repeated five times. CsCl density gradient centrifugation. Analytical CsCl centrifugation was carried out in a Spinco model E ultracentrifuge at 44,770 rpm for 20 h at 250C as described by Nagley and Linnane (18). Preparative CsCl gradients used for the separation of nuclear and mitochondrial DNA were run at 35,000 rpm and 200C for 65 h in an MSE fixed-angle 10 x 10 ml rotor (Measuring & Scientific Equipment, Ltd., London). CsCl solutions were 5.0 ml in volume and contained 55.7% CsCl (by weight) in 10 mM Tris, 1 mM EDTA, pH 6.8, at a starting density of 1.691 g/ml. Whole-cell yeast DNA, labeled with [14C]adenine in vivo and extracted by the procedure of Nagley and Linnane (18), was used as a density marker. Preparative neutral CsCl gradients for the analysis of mtDNA density labeled with 5-bromodeoxyuridine were centrifuged at 28,000 rpm and 250C for 65 h in a 3 x 5 ml MSE swing-out rotor. DNA was in a 4.5-ml CsCl solution in 10 mM Tris, 1 mM EDTA,

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pH 6.8, at a density of 1.738 g/ml (58.0% CsCl by weight). E. coli [14C]DNA was added as a density marker. Alkaline CsCl gradients, for the determination of the single-stranded density of bromouracil-labeled DNA, were centrifuged at 31,000 rpm and 250C for 65 h in a Spinco 40 fixed-angle rotor. CsCl solutions were 5.0 ml in volume and contained 62.4% CsCl (by weight) in 0.05 M NaOH, pH approximately 12.5. The starting density was 1.84 g/ml. Fractions (0.1 ml) were collected from the bottom of the tube, using a paraffin oil overlay method. Fractions from gradients containing marker [14C]DNA were treated with 0.5 M NaOH at 300C for 16 h to hydrolyze RNA. DNA was co-precipitated with bovine serum albumin (0.2 mg) and calf thymus DNA (0.2 mg) by the addition of ice-cold 20% trichloroacetic acid, to a final concentration of 7%, and then collected on GF/C glass fiber filters. Filters were washed twice with 10 ml of 5% trichloroacetic acid and once with water and then dried. Precipitates were solubilized and radioactivity was counted as described above. To determine densities (p0.25') at different places in the gradient, 10 ,ul was withdrawn from 0. 1-ml fractions immediately after collection and the refractive index at 25°C (inD25°) was measured with an Abbe Refractometer (Atago). Density was calculated from refractive index using the relationship p0.251 = 10.8601 'RD25 - 13.9474, valid for the density range 1.2 to 1.9 g/ml (26). Sucrose gradient sedimentation. For sucrose gradient rate sedimentation, 0.2 ml of DNA in 10 mM Tris-hydrochloride, 1 mM EDTA (pH 8.0) was layered onto a preformed 5 to 20% sucrose (wt/vol) gradient (6 ml) containing 10 mM Tris, 1 mM EDTA (pH 6.8). Centrifugation was carried out at 200C in an MSE 3 x 6.5 ml swing-out rotor for 2 h at 50,000 rpm. Fractions (0.2 ml) were collected from the bottom of the tube, and DNA was precipitated and collected on glass fiber filters as described above. Yeast nuclear [14C]DNA was added as a marker, and molecular weight calculations were based on its having a molecular weight of 16;6 x 106. The bulk of the marker DNA sedimented as a fairly homogenous peak. Its molecular weight was derived as follows: the S value was determined from its sedimentation rate (in 1.0 M NaCl) in a Spinco model E ultracentrifuge and converted to s20,w (25). The molecular weight (M) was calculated from the equation: S20 w= 2.8 + 0.00834, M0-479 (7).

RESULTS Characteristics of DNA synthesis in isolated mitochondria. Mitochondria isolated from strain L411 were incubated at 28°C in the presence of the four deoxyribonucleotide triphosphates, ATP, and an ATP-regenerating system. The kinetics of incorporation of [3H]dITP into alkali-stable, acid-precipitable material is shown in Fig. 1. The rate of incorporation of dTTP is approximately linear for 20

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