Eur. J . Biochem. 68, 199-207 (1976)
DNA-Dependent DNA Polymerase from Yeast Mitochondria Dependence of Enzyme Activity on Conditions of Cell Growth, and Properties of the Highly Purified Polymerase Ulrike WINTERSBERGER and Hans BLUTSCH Institut fiir Krebsforschung der Univcrsitiit Wicn (Received March 16IJuly 2, 1976)
The activity of DNA polymerase was determined in gradient-purified mitochondria from yeast cells grown under a variety of conditions. The specific enzyme activity was found to be dependent on the degree of aeration of the cells, and on tlie carbon source used for the medium. It was sensitive to glucose repression, and was enhanced about two-fold by the growth of yeast cells in the presence of ethidium bromide. Mitochondrial DNA polymerase was highly purified and several properties were determined. Sucrose density gradient centrifugation, and dodecylsulfate-polyacrylamide gel electrophoresis revealed tlie following structure : a monomer of molecular weight around 60000 aggregated under relatively high salt concentration (0.2 M phosphate buffer) to a dimer of about 120000 which under low salt concentration (0.02 M Tris-HC1 buffer) formed higher aggregates. For optimal activity an M$+ ion concentration of 50 inM was found necessary, Mn ions did not promote activity at any concentration tested (0.5- 50 mM). Indeed, if added to M$+-containing assays, Mn2+ strongly inhibited enzyme activity at low concentrations. This might be an explanation for the inducation of mitochondrial mutants in yeast cells grown in the presence of Mn2' ions. Mitochondrial DNA polymerase activity was strongly inhibited by low concentrations of the - SH reagent p-chloromercuribenzoate, the nucleotide analogue cytosine arabinoside triphosphate also exerted an inhibitory effect. An about 50'%, decrease of activity was observed in the presence of 1 mM o-phenanthroline in assay mixture containing DNA at about the K, comentration. The enzyme preferred a gapped template primer, poly(dA) . (dT),,, over nicked DNA and was unable to use a polyribonucleotide template, poly(rt?) . (dT),, . In the purest preparations no exonuclease activity could be detected At least two DNA-synthesizing systems are operative in a eukaryotic cell: one in the nucleus, the other in the mitochondria. The recent progress concerning our knowledge of the mechanism of mitochondrial DNA synthesis [l -31 as well as the idea that the DNA-synthesizing apparatus of the organelles might perhaps be simpler and easier to investigate than that of the nucleus, prompted us to start isolating the components of the mitochondrial DNA-synthesizing inachinery. In an earlier publication [4] we had already described the properties of a partially purified preparation of DNA polymerase from yeast mitochondria Enzjiiies. D N A polymerase or deoxynucleosidc triphosphate : DNA deoxynucleotidyltransferase (EC 2.7.7.7) ; alcohol dehydrogenase or alcohol : NAD oxidoreductase (EC 1.1.1.1); malate dehydrogenase or 1.-malate : N A D oxidoreductase (EC 1.1.1.37); RNA polymcrasc or nucleosidetriphosphate : RNA nucleotidyl-
transferase (EC 2.7.7.6).
and had shown that this enzyme was most probably coded for by the nuclear genome. Comparably pure enzyme preparations of mitochondrial DNA polymerases were studied from rat liver [5,6], HeLa cells [7,8] and Tetrahymena [9]. In this paper we want to summarize the results of our continued studies of the dependence of enzyme activity on growth conditions, the improvement of the purification procedure and some properties of the highly purified mitochondrial DNA polymerase. EXPERIMENTAL PROCEDURE Yeast Strain and Gmt>tlzof Cells The diploid laboratory strain, A-1 160 of Succharornyces cerevisiue, was grown at 30 "C in a New Brunswick fermentator FS 314. The medium contained 0.3%, yeast extract (Difco), 0.5x bacto peptone
200
(Difco), and glucose, galactose or glycerol (at concentrations indicated for the individual experiments) as carbon sources.
Bzdfev Buffer A contained 0.02 M Tris-HC1, pH 7.5, 1 mM EDTA, 1 mM dithioerythritol, and 6 mg/l of the protease inhibitor phenylmethylsulfonyl fluoride. Assuys The standard assay for DNA polymerase activity was carried out as previously described [lo, 1I] with salmon sperm DNA activated according to Aposhian and Kornberg [I21 as template primer. One enzyme unit incorporates 1 nmol of dTMP in 15 min at 35 "C. The test mixture for exonuclease activity contained 50 mM Tris-HCI (pH 7.5), 10 mM MgCI,, 1 mg/ml bovine serum albumin, 15 pg [3H]DNA from Eschrrichiu coli (40000 counts/min, native or activated with pancratic deoxyribonuclease) and enzyme protein in a total volume of 650 pl. After 0, 5, 10, 30, 60 and 120 min of incubation at 35 "C, 100-p1 aliquots were removed and pipetted onto Whatman GF/C filters. These were dropped into lo"/, trichloroacetic acid to precipitate DNA and then were washed extensively with 5% trichloroacetic acid and finally with ethanol/ ether (l/l).The filters were dried and the radioactivity of residual high-molecular-weight DNA was determined. Alcohol dehydrogenase and malate dehydrogenase activities were determined as outlined in the Boehringer Information booklet (1973), protein content was measured by the Lowry method 1131, DNA was determined with diphenylamine [14], and RNA by the orcinol method [15]. Purification of Mitochndrial DNA Polq'nzeruse All operations were carried out at 0-4 "C. Mitochondria were prepared as described earlier [4] except that for the final purification 20 - 70"/, sucrose gradients in buffer A were used instead of Urographin gradients. The initial steps of the preparation procedure for DNA polymerase from purified mitochondria were in principle similar to those published earlier [4]. As some modifications proved advantageous and further steps were added, the whole improved procedure is briefly described as follows. Mitochondria were suspended in buffer A to about 12 mg protein/ ml and were homogenized by treatment with an UltraTurrax homogenizer (Janke and Kunkel, Germany) for 20 s. Sodium cloride was added to a final concentration of 0.5 M and the Ultra-Turrax treatment was repeated three more times with extensive cooling during the intervals. After centrifugation for 30 min
DNA-Dependent DNA Polymerase from Yeast Mitochondria
at 20000g, the clear supernatant was dialyzed for 2 h against 20 vol. of buffer A containing 0.1 M NaCl and 10% glycerol. The mitochondria1 extract was applied to a DEAE-cellulose column previously equilibrated with the dialysis buffer. The size of the column was chosen so that 1 ml of column volume corresponded to 10 mg of protein applied. The column was washed with buffer A containing 0.2 M NaCl and 10% glycerol until no more protein could be eluted. Three column volumes of a gradient of 0.2-0.4 M NaCl in buffer A (10% glycerol) were then pumped through the column, which was finally washed with about one column volume of buffer A containing 0.4 NaCI. DNA polymerase activity was determined in aliquots of the eluted fractions. Active ones were combined, dialyzed against buffer A containing 0.1 M NaCl and 10%) glycerol, and were adsorbed onto a DNA-cellulose column [16] previously equilibrated with the same buffer. A column volume of 1 ml was used per mg of enzyme protein. Elution was carried out stepwise with buffer A containing increasing amounts of NaCl (0.1 M, 0.2 M, 0.6 M). The active enzyme was recovered with the fraction containing 0.6 M NaCl and for storage was dialyzed against buffer A with 50% glycerol. Sucrose gradients were run for further purification as well as for the determination of the molecular weight of DNA polymerase. For this purpose the enzyme solution was dialyzed for 3 h against 0.2 M potassium phosphate buffer (pH 7.4), and, in case of gradients for molecular weight determination, it was mixed with alcohol dehydrogenase (1.5 mg/ml solution) and malate dehydrogenase (0.1 mg/ml solution) as marker enzymes. 0.5 ml each were layered onto 11.5 ml of a 5 - 20% sucrose gradient in 0.2 M potassium phosphate buffer and centrifuged in the SW 41 rotor of a Beckmann ultracentrifuge for 16 h at 40000 rev./min. After fractionation, activities of DNA polymerase as well as of alcohol and malate dehydrogenase were determined. Fractions containing DNA polymerase from gradients centrifuged without marker enzymes were pooled and dialyzed against buffer A containing 50"/, glycerol. Enzyme solutions could be kept in liquid nitrogen for several months without significant loss of activity. Once warmed up to 0 "C, however, enzyme activity decreased rapidly.
Dodecylsulfirte- Polq.ucrylumide Gel Elrctrophoresis
Electrophoresis was carried out according to Weber and Osborn [17] with samples containing 10-50 pg of protein, 100 p1 of buffer (0.05 M sodium phosphate pH 7.1, 0.5% sodium dodecylsulfate, 5% 2-mercaptoethanol) and 40% glycerol in a final volume of 500 pl. After heating for 3 min at 100 "C (boiling water bath) samples were applied to 7.5% gels and
U . Wintersbcrger and H. Blutsch
electrophoresis was carried out for 4 h at 8 m A per gel.
Clzenzicals Deoxyribonucleotide triphosphates, cytosine arabinoside triphosphate and salmon sperm DNA (type 111) were from Sigma Chemical Co (U.S.A.), tritiated thymidine triphosphate and deoxyguanosine tripliosphate were purchased from the Radiochemical Centre (Amersham, England), poly(dA) (dT),, , poly(rA) . (dT),,, poly(dC) and (dG),, were obtained from P. L. Laboratories (U.S.A.), and poly[d(A-T)] from Miles Laboratories. Alcohol dehydrogenase, malate dehydrogenase and E. coli RNA polymerase came from Boehringer (Mannheim, Germany), pancreatic ribonuclease from Worthington Biochem. Corp. (U.S.A.), DEAE-cellulose was from Serva (Heidelberg, Germany) and DNA-cellulose was prepared as described elsewhere [16]. All other chemicals were of analytical quality.
RESULTS AND DISCUSSION
Dependence of Mitochondria1 D N A Polymeruse Activi fy on the Growth Conditions of the Cells Yeast cells as facultative anaerobes are able to change significantly the enzymic equipment of their mitochondria according to the quantity of oxygen available. In addition, amount and structure of mitochondria varies with the carbon source and is sensitive to glucose repression. We were interested to learn whether the activity of mitochondrial DNA polymerase, an enzyme coded for by the nuclear genome, was regulated by the supply of oxygen and glucose or whether this enzyme was produced constitutively. Yeast cells were grown under varying conditions and besides the general parameters of yield and cell number, the amount of mitochondrial protein per gram of cells (wet weight), specific DNA polymerase activity of mitochondria as well as activity in reference to whole cell mass were determined. The results are summarized in Table 1. From the first five lines the influence of an increase of aeration on mitochondrial DNA polymerase from stationary phase cells grown in presence of low glucose concentration can be seen. Zero aeration (Expt 1) does not, of course, correspond to anaerobic growth since by stirring the medium in the open system of the fermentor the cells always obtain a certain amount of oxygen. Nevertheless, only low quantities of mitochondrial protein can be isolated from these cells. With increasing amounts of air pumped through the medium, not only the mitochondrial mass but also the content of DNA polymerase activity in the organelles rises appreciably. More specifically, a three-fold increase of mitochon-
20 1
drial protein is accompanied by a five-fold rise i n specific activity leading to about 15 times more mitochondrial DNA polymerase activity in cells grown with high aeration (18 Ijmin) compared to the nonaerated ones. An increase of the glucose concentration from 0.8x to 5% (Expt 6) leads to a much higher cell yield, whereas mitochondrial protein per gram cells and specific activity of DNA polymerase drop considerably. If glucose-grown cells are harvested during the late logarithmic phase (Expts 8 - 10) a lower specific activity of initochondrial DNA polymerase is observed. This indicates an induction of the enzyme while glucose is consumed by the cells. From galactosegrown cells, in contrast, a similar amount of total mitochondrial DNA polymerase activity per gram cells can be isolated independently whether cells are harvested during the exponential phase or during the stationary phase of growth (Expts 7 and 1I ) , since no glucose repression is operative in these cells. As expected, the highest amount of mitochondria1 protein and of total activity of mitochondrial DNA polymerase per cell is found in cultures.from a medium containing glycerol as carbon source (Expt 12). The specific DNA polymerase activity of these mitochondria does not, however, exceed that from fully induced glucose-grown cells (compare Expt 12 to Expts 4 and 5). From these results we conclude that the activity of mitochondrial DNA polymerase is regulated similarly to other enzymes of the organelles, especially those concerned with respiration. Our findings correspond to earlier reports on enhanced synthesis of mitochondrial DNA during oxygen induction [I81 or during release from glucose repression [19]. They show that this increased DNA synthesis is managed by the ccll by raising the mitochondrial DNA polymerase activity, and they also can be taken as an argument against the suspicion [I] that the enzyme might be only involved in repair synthesis and not in DNA replication. It is well known that the intercalating dye ethidium bromide severely interferes with mitochondrial DNA synthesis and it has been reported by Westergaard et al. [20]that in Tetrahymena, probably as a compensation against loss of DNA, mitochondrial DNA polymerase activity is enhanced by the drug. Therefore we measured DNA polymerase in cells grown in the presence of ethidium bromide. In the first experiment 10 mg (Expt 13) or 50 mg (Expt 14) of the dye per I medium were added for the last 5 11 of growth. An about 30-40%, enhancement of the specific activity of the intact mitochondria and a more than twofold rise of that of the mitochondrial extract was observed. Although this increase was much lower than that reported for Tetruhymena [20], it nevertheless may represent a certain compensatory reaction of the cell to the damage caused by ethidium bromide. If the drug is added at the beginning of the growth period
202
DNA-Dependent D N A Polymerase from Yeast Mitochondria
Table 1, Depnc/ence of nzitoclwndrial D N A poljmera.se uctiuity on the growth conditions of fhe cell.\ It should be noted that the accuracy of the numbers for mitochondrial protein yield is limited by the fact that the harvest of the mitochondrial band from the gradient was carried out manually and was controlled by observation with the eye only. DNA polymerase activity was determined as described in Experimental Procedure. Each assay contained between 40 - 50 big of protein. EtdBr = ethidium bromide Expt
Growth conditions
Yield -~
~
DNA polymerase activity in mitochondrial suspension _ _ ~ .~ total specific activity activity
carbon source
time
aeration
wet weight
xceli number
mitochondrial protein
'%,
h
llmin
Sil
ml-'
mglg cells
unitslg cells
units/mg protein
1
Glucose 08
14
0
5.6
2.8
0.7
0.037
0.054
2
Glucose 0.8
14
6
7.5
7.0
0.8
0.063
0.077
3
Glucose 08
14
11
10.4
7.6
0.9
0.364
0.270
4
Glucose 0.8
14
16
11.7
9.0
1.6
0.487
0.309
5
Glucose 0.8
14
18
12.7
13.0
2.2
0.591
0.272
6
Glucose 5
14
18
24.6
19.0
0.9
0.177
0.202
7
Galactose 3
14
18
15.4
18.0
1.4
0.413
0.302
8
Glucose 0.8
10
18
8.5
5.5
1.8
0.223
0.111
9
Glucose 8
10
18
18.8
9.0
1.0
0.085
0.086
10
Glucose 8
10
0
11.6
5.4
0.6
0.047
0.086
11
Galactose
10
18
4.7
3.0
2.0
0.403
0.206
Glycerol
10
18
1.8
1.4
3.7
1.030
0.278
12
4
13
Glucose 0.x (5 h 30 mgll EtdBr)
14
18
11.8
12.0
2.2
0.863
0.386
14
Glucose 0.8 ( 5 h 50 ingll EtdRr)
14
18
10.5
6.4
1 .x
0.775
0.425
15
Glucose 0.8 (14 h 5 mgll EtdBr)
14
18
6.8
4.5
0.5
0.216
0.428
( 5 mg/l medium, expt 15) all cells are converted to petite mutants. Mitochondria1 protein is decreased to about one quarter of that of untreated cells, whereas specific activity of DNA polymerase of the few mitochondria left is still high.
Purification Procedure jor the Mitocl~onu'riulDNA Po(i*nrerusefrom Yeast If yeast cells are fractionated three DNA-dependent DNA polymerases can be obtained : two,
enzymes A and B, from the nuclei or the cytoplasm [lo, 11,211 and a third from mitochondria. It seems worth noting that whereas the nuclear enzymes are bound only weakly to the nuclei and in fact are therefore found to a great extent in the cytoplasm of fractionated cells [lo, 111, the mitochondrial enzyme on the other hand is tightly bound to the mitochondrial membrane and can only be extracted by buffers containing high salt concentrations and employing extensive mechanical disruption. This is reininiscent of another mitochondrial enzyme of macromolecular
U. Wintersbcrger and H. Blutsch
201
Table 2. Purificution nJ niitochondviul D N A po1~~merrr.w j?oni y u s i Yeast cells were grown for 14 h at maximal aeration in a medium containing 0.3'>. A. (1971) Riochwi. Biopti1,s. Rrs. f'onzinzrn. 44, 37 -43. 29. Springgate, C . F., Mildvan, A . S., Ahramson, R., Engle, J. L. & Locb, L. A. (1973) J . Biol. C'ht~iw. 248, 5987-5993. 30. Poiesz, B. J.. Seal, G. & Loeb, L. A . (1974) Pro(,. Ntrtl A m / . S(,i. C:.S.A. 71. 4892-4896. 31, Rama Reddy, G. V., Goulian, M. 81 Hendlci-. S. S . (1971) Nor. New Biol. 234, 286-288. 32. Momparler, R. L., Rossi, M. & Lahivan. A. (1973)J. Biol. Clrcni. 248,285-293. 33. Lynch, W. E. & Lieberman, J. (1973) Bioc,/rrni. Uiopl7j~.s.Rc.t. Corwnirn. 52, 843 - 849. -
REFERENCES 1. Borst, P. (1972) Annu. Rev. Bioclien?. 41, 333-376. _. 7 Kasamatsu, H. & Vinograd. J . (1974) Annu. Rea. Biochrm. 43, 695-719. 3. Wintersberger, E. (1974) Curr. Top. Microhiol. 6 6 , 77- 102. 4. Wintersberger, U. & Wintersberger, E. (1970) Eur. J . Biockt'fT7. 13. 20 - 27. 5. Kalf, F. G. & Ch'ih J. J. (1968) J . Biol. Chon. 243, 4904-4916. 6 . Mrqcr. R . R . B Simpson. M . V. (1970) .1. Bwl. ('hcvn. 245. 3426 - 3435. 7. 'ribbeits, C. J. 8. & Vinograd. J. (1973) J . Biol. Chcw?. 248, 3367 - 3379. 8. Fry, M. & Weissbach, A. (1973) Biocherni.s1r4; 12, 3602-3608. 9. Westergaard. 0. & Lindberg, B. (1972) Eur. J . Biochrnr. ZX, 422-431. 10. Wintersberger. U. & Wintersberger, E. (1970) Eur. J . Biod7er77. 13, 11-19. 11. Wintersbcrger, E. (1974) Eur. J . Biochern. 50, 41 -47. 12. Aposbian, H. V. & Kornberg, A. (1962) J . Biol. C h n . 237. 519 - 525. 13. Lowry. 0. M., Kosebrough, N . J., Farr, A. L. & Randall. R . J . (1951) .I. Bid. C h m . 193, 265-275.
U. Wintcrsberger and H. Blutsch, Institut f i r Krehsfoi-schung der Universitit Wien, Borschkegasse 8a. A-1090 Wien, Austria
DNA-Dependent DNA Polymerase from Yeast Mitochondria Dependence of Enzyme Activity on Conditions of Cell Growth, and Properties of the Highly Purified Polymerase Ulrike WINTERSBERGER and Hans BLUTSCH Institut fiir Krebsforschung der Univcrsitiit Wicn (Received March 16IJuly 2, 1976)
The activity of DNA polymerase was determined in gradient-purified mitochondria from yeast cells grown under a variety of conditions. The specific enzyme activity was found to be dependent on the degree of aeration of the cells, and on tlie carbon source used for the medium. It was sensitive to glucose repression, and was enhanced about two-fold by the growth of yeast cells in the presence of ethidium bromide. Mitochondrial DNA polymerase was highly purified and several properties were determined. Sucrose density gradient centrifugation, and dodecylsulfate-polyacrylamide gel electrophoresis revealed tlie following structure : a monomer of molecular weight around 60000 aggregated under relatively high salt concentration (0.2 M phosphate buffer) to a dimer of about 120000 which under low salt concentration (0.02 M Tris-HC1 buffer) formed higher aggregates. For optimal activity an M$+ ion concentration of 50 inM was found necessary, Mn ions did not promote activity at any concentration tested (0.5- 50 mM). Indeed, if added to M$+-containing assays, Mn2+ strongly inhibited enzyme activity at low concentrations. This might be an explanation for the inducation of mitochondrial mutants in yeast cells grown in the presence of Mn2' ions. Mitochondrial DNA polymerase activity was strongly inhibited by low concentrations of the - SH reagent p-chloromercuribenzoate, the nucleotide analogue cytosine arabinoside triphosphate also exerted an inhibitory effect. An about 50'%, decrease of activity was observed in the presence of 1 mM o-phenanthroline in assay mixture containing DNA at about the K, comentration. The enzyme preferred a gapped template primer, poly(dA) . (dT),,, over nicked DNA and was unable to use a polyribonucleotide template, poly(rt?) . (dT),, . In the purest preparations no exonuclease activity could be detected At least two DNA-synthesizing systems are operative in a eukaryotic cell: one in the nucleus, the other in the mitochondria. The recent progress concerning our knowledge of the mechanism of mitochondrial DNA synthesis [l -31 as well as the idea that the DNA-synthesizing apparatus of the organelles might perhaps be simpler and easier to investigate than that of the nucleus, prompted us to start isolating the components of the mitochondrial DNA-synthesizing inachinery. In an earlier publication [4] we had already described the properties of a partially purified preparation of DNA polymerase from yeast mitochondria Enzjiiies. D N A polymerase or deoxynucleosidc triphosphate : DNA deoxynucleotidyltransferase (EC 2.7.7.7) ; alcohol dehydrogenase or alcohol : NAD oxidoreductase (EC 1.1.1.1); malate dehydrogenase or 1.-malate : N A D oxidoreductase (EC 1.1.1.37); RNA polymcrasc or nucleosidetriphosphate : RNA nucleotidyl-
transferase (EC 2.7.7.6).
and had shown that this enzyme was most probably coded for by the nuclear genome. Comparably pure enzyme preparations of mitochondrial DNA polymerases were studied from rat liver [5,6], HeLa cells [7,8] and Tetrahymena [9]. In this paper we want to summarize the results of our continued studies of the dependence of enzyme activity on growth conditions, the improvement of the purification procedure and some properties of the highly purified mitochondrial DNA polymerase. EXPERIMENTAL PROCEDURE Yeast Strain and Gmt>tlzof Cells The diploid laboratory strain, A-1 160 of Succharornyces cerevisiue, was grown at 30 "C in a New Brunswick fermentator FS 314. The medium contained 0.3%, yeast extract (Difco), 0.5x bacto peptone
200
(Difco), and glucose, galactose or glycerol (at concentrations indicated for the individual experiments) as carbon sources.
Bzdfev Buffer A contained 0.02 M Tris-HC1, pH 7.5, 1 mM EDTA, 1 mM dithioerythritol, and 6 mg/l of the protease inhibitor phenylmethylsulfonyl fluoride. Assuys The standard assay for DNA polymerase activity was carried out as previously described [lo, 1I] with salmon sperm DNA activated according to Aposhian and Kornberg [I21 as template primer. One enzyme unit incorporates 1 nmol of dTMP in 15 min at 35 "C. The test mixture for exonuclease activity contained 50 mM Tris-HCI (pH 7.5), 10 mM MgCI,, 1 mg/ml bovine serum albumin, 15 pg [3H]DNA from Eschrrichiu coli (40000 counts/min, native or activated with pancratic deoxyribonuclease) and enzyme protein in a total volume of 650 pl. After 0, 5, 10, 30, 60 and 120 min of incubation at 35 "C, 100-p1 aliquots were removed and pipetted onto Whatman GF/C filters. These were dropped into lo"/, trichloroacetic acid to precipitate DNA and then were washed extensively with 5% trichloroacetic acid and finally with ethanol/ ether (l/l).The filters were dried and the radioactivity of residual high-molecular-weight DNA was determined. Alcohol dehydrogenase and malate dehydrogenase activities were determined as outlined in the Boehringer Information booklet (1973), protein content was measured by the Lowry method 1131, DNA was determined with diphenylamine [14], and RNA by the orcinol method [15]. Purification of Mitochndrial DNA Polq'nzeruse All operations were carried out at 0-4 "C. Mitochondria were prepared as described earlier [4] except that for the final purification 20 - 70"/, sucrose gradients in buffer A were used instead of Urographin gradients. The initial steps of the preparation procedure for DNA polymerase from purified mitochondria were in principle similar to those published earlier [4]. As some modifications proved advantageous and further steps were added, the whole improved procedure is briefly described as follows. Mitochondria were suspended in buffer A to about 12 mg protein/ ml and were homogenized by treatment with an UltraTurrax homogenizer (Janke and Kunkel, Germany) for 20 s. Sodium cloride was added to a final concentration of 0.5 M and the Ultra-Turrax treatment was repeated three more times with extensive cooling during the intervals. After centrifugation for 30 min
DNA-Dependent DNA Polymerase from Yeast Mitochondria
at 20000g, the clear supernatant was dialyzed for 2 h against 20 vol. of buffer A containing 0.1 M NaCl and 10% glycerol. The mitochondria1 extract was applied to a DEAE-cellulose column previously equilibrated with the dialysis buffer. The size of the column was chosen so that 1 ml of column volume corresponded to 10 mg of protein applied. The column was washed with buffer A containing 0.2 M NaCl and 10% glycerol until no more protein could be eluted. Three column volumes of a gradient of 0.2-0.4 M NaCl in buffer A (10% glycerol) were then pumped through the column, which was finally washed with about one column volume of buffer A containing 0.4 NaCI. DNA polymerase activity was determined in aliquots of the eluted fractions. Active ones were combined, dialyzed against buffer A containing 0.1 M NaCl and 10%) glycerol, and were adsorbed onto a DNA-cellulose column [16] previously equilibrated with the same buffer. A column volume of 1 ml was used per mg of enzyme protein. Elution was carried out stepwise with buffer A containing increasing amounts of NaCl (0.1 M, 0.2 M, 0.6 M). The active enzyme was recovered with the fraction containing 0.6 M NaCl and for storage was dialyzed against buffer A with 50% glycerol. Sucrose gradients were run for further purification as well as for the determination of the molecular weight of DNA polymerase. For this purpose the enzyme solution was dialyzed for 3 h against 0.2 M potassium phosphate buffer (pH 7.4), and, in case of gradients for molecular weight determination, it was mixed with alcohol dehydrogenase (1.5 mg/ml solution) and malate dehydrogenase (0.1 mg/ml solution) as marker enzymes. 0.5 ml each were layered onto 11.5 ml of a 5 - 20% sucrose gradient in 0.2 M potassium phosphate buffer and centrifuged in the SW 41 rotor of a Beckmann ultracentrifuge for 16 h at 40000 rev./min. After fractionation, activities of DNA polymerase as well as of alcohol and malate dehydrogenase were determined. Fractions containing DNA polymerase from gradients centrifuged without marker enzymes were pooled and dialyzed against buffer A containing 50"/, glycerol. Enzyme solutions could be kept in liquid nitrogen for several months without significant loss of activity. Once warmed up to 0 "C, however, enzyme activity decreased rapidly.
Dodecylsulfirte- Polq.ucrylumide Gel Elrctrophoresis
Electrophoresis was carried out according to Weber and Osborn [17] with samples containing 10-50 pg of protein, 100 p1 of buffer (0.05 M sodium phosphate pH 7.1, 0.5% sodium dodecylsulfate, 5% 2-mercaptoethanol) and 40% glycerol in a final volume of 500 pl. After heating for 3 min at 100 "C (boiling water bath) samples were applied to 7.5% gels and
U . Wintersbcrger and H. Blutsch
electrophoresis was carried out for 4 h at 8 m A per gel.
Clzenzicals Deoxyribonucleotide triphosphates, cytosine arabinoside triphosphate and salmon sperm DNA (type 111) were from Sigma Chemical Co (U.S.A.), tritiated thymidine triphosphate and deoxyguanosine tripliosphate were purchased from the Radiochemical Centre (Amersham, England), poly(dA) (dT),, , poly(rA) . (dT),,, poly(dC) and (dG),, were obtained from P. L. Laboratories (U.S.A.), and poly[d(A-T)] from Miles Laboratories. Alcohol dehydrogenase, malate dehydrogenase and E. coli RNA polymerase came from Boehringer (Mannheim, Germany), pancreatic ribonuclease from Worthington Biochem. Corp. (U.S.A.), DEAE-cellulose was from Serva (Heidelberg, Germany) and DNA-cellulose was prepared as described elsewhere [16]. All other chemicals were of analytical quality.
RESULTS AND DISCUSSION
Dependence of Mitochondria1 D N A Polymeruse Activi fy on the Growth Conditions of the Cells Yeast cells as facultative anaerobes are able to change significantly the enzymic equipment of their mitochondria according to the quantity of oxygen available. In addition, amount and structure of mitochondria varies with the carbon source and is sensitive to glucose repression. We were interested to learn whether the activity of mitochondrial DNA polymerase, an enzyme coded for by the nuclear genome, was regulated by the supply of oxygen and glucose or whether this enzyme was produced constitutively. Yeast cells were grown under varying conditions and besides the general parameters of yield and cell number, the amount of mitochondrial protein per gram of cells (wet weight), specific DNA polymerase activity of mitochondria as well as activity in reference to whole cell mass were determined. The results are summarized in Table 1. From the first five lines the influence of an increase of aeration on mitochondrial DNA polymerase from stationary phase cells grown in presence of low glucose concentration can be seen. Zero aeration (Expt 1) does not, of course, correspond to anaerobic growth since by stirring the medium in the open system of the fermentor the cells always obtain a certain amount of oxygen. Nevertheless, only low quantities of mitochondrial protein can be isolated from these cells. With increasing amounts of air pumped through the medium, not only the mitochondrial mass but also the content of DNA polymerase activity in the organelles rises appreciably. More specifically, a three-fold increase of mitochon-
20 1
drial protein is accompanied by a five-fold rise i n specific activity leading to about 15 times more mitochondrial DNA polymerase activity in cells grown with high aeration (18 Ijmin) compared to the nonaerated ones. An increase of the glucose concentration from 0.8x to 5% (Expt 6) leads to a much higher cell yield, whereas mitochondrial protein per gram cells and specific activity of DNA polymerase drop considerably. If glucose-grown cells are harvested during the late logarithmic phase (Expts 8 - 10) a lower specific activity of initochondrial DNA polymerase is observed. This indicates an induction of the enzyme while glucose is consumed by the cells. From galactosegrown cells, in contrast, a similar amount of total mitochondrial DNA polymerase activity per gram cells can be isolated independently whether cells are harvested during the exponential phase or during the stationary phase of growth (Expts 7 and 1I ) , since no glucose repression is operative in these cells. As expected, the highest amount of mitochondria1 protein and of total activity of mitochondrial DNA polymerase per cell is found in cultures.from a medium containing glycerol as carbon source (Expt 12). The specific DNA polymerase activity of these mitochondria does not, however, exceed that from fully induced glucose-grown cells (compare Expt 12 to Expts 4 and 5). From these results we conclude that the activity of mitochondrial DNA polymerase is regulated similarly to other enzymes of the organelles, especially those concerned with respiration. Our findings correspond to earlier reports on enhanced synthesis of mitochondrial DNA during oxygen induction [I81 or during release from glucose repression [19]. They show that this increased DNA synthesis is managed by the ccll by raising the mitochondrial DNA polymerase activity, and they also can be taken as an argument against the suspicion [I] that the enzyme might be only involved in repair synthesis and not in DNA replication. It is well known that the intercalating dye ethidium bromide severely interferes with mitochondrial DNA synthesis and it has been reported by Westergaard et al. [20]that in Tetrahymena, probably as a compensation against loss of DNA, mitochondrial DNA polymerase activity is enhanced by the drug. Therefore we measured DNA polymerase in cells grown in the presence of ethidium bromide. In the first experiment 10 mg (Expt 13) or 50 mg (Expt 14) of the dye per I medium were added for the last 5 11 of growth. An about 30-40%, enhancement of the specific activity of the intact mitochondria and a more than twofold rise of that of the mitochondrial extract was observed. Although this increase was much lower than that reported for Tetruhymena [20], it nevertheless may represent a certain compensatory reaction of the cell to the damage caused by ethidium bromide. If the drug is added at the beginning of the growth period
202
DNA-Dependent D N A Polymerase from Yeast Mitochondria
Table 1, Depnc/ence of nzitoclwndrial D N A poljmera.se uctiuity on the growth conditions of fhe cell.\ It should be noted that the accuracy of the numbers for mitochondrial protein yield is limited by the fact that the harvest of the mitochondrial band from the gradient was carried out manually and was controlled by observation with the eye only. DNA polymerase activity was determined as described in Experimental Procedure. Each assay contained between 40 - 50 big of protein. EtdBr = ethidium bromide Expt
Growth conditions
Yield -~
~
DNA polymerase activity in mitochondrial suspension _ _ ~ .~ total specific activity activity
carbon source
time
aeration
wet weight
xceli number
mitochondrial protein
'%,
h
llmin
Sil
ml-'
mglg cells
unitslg cells
units/mg protein
1
Glucose 08
14
0
5.6
2.8
0.7
0.037
0.054
2
Glucose 0.8
14
6
7.5
7.0
0.8
0.063
0.077
3
Glucose 08
14
11
10.4
7.6
0.9
0.364
0.270
4
Glucose 0.8
14
16
11.7
9.0
1.6
0.487
0.309
5
Glucose 0.8
14
18
12.7
13.0
2.2
0.591
0.272
6
Glucose 5
14
18
24.6
19.0
0.9
0.177
0.202
7
Galactose 3
14
18
15.4
18.0
1.4
0.413
0.302
8
Glucose 0.8
10
18
8.5
5.5
1.8
0.223
0.111
9
Glucose 8
10
18
18.8
9.0
1.0
0.085
0.086
10
Glucose 8
10
0
11.6
5.4
0.6
0.047
0.086
11
Galactose
10
18
4.7
3.0
2.0
0.403
0.206
Glycerol
10
18
1.8
1.4
3.7
1.030
0.278
12
4
13
Glucose 0.x (5 h 30 mgll EtdBr)
14
18
11.8
12.0
2.2
0.863
0.386
14
Glucose 0.8 ( 5 h 50 ingll EtdRr)
14
18
10.5
6.4
1 .x
0.775
0.425
15
Glucose 0.8 (14 h 5 mgll EtdBr)
14
18
6.8
4.5
0.5
0.216
0.428
( 5 mg/l medium, expt 15) all cells are converted to petite mutants. Mitochondria1 protein is decreased to about one quarter of that of untreated cells, whereas specific activity of DNA polymerase of the few mitochondria left is still high.
Purification Procedure jor the Mitocl~onu'riulDNA Po(i*nrerusefrom Yeast If yeast cells are fractionated three DNA-dependent DNA polymerases can be obtained : two,
enzymes A and B, from the nuclei or the cytoplasm [lo, 11,211 and a third from mitochondria. It seems worth noting that whereas the nuclear enzymes are bound only weakly to the nuclei and in fact are therefore found to a great extent in the cytoplasm of fractionated cells [lo, 111, the mitochondrial enzyme on the other hand is tightly bound to the mitochondrial membrane and can only be extracted by buffers containing high salt concentrations and employing extensive mechanical disruption. This is reininiscent of another mitochondrial enzyme of macromolecular
U. Wintersbcrger and H. Blutsch
201
Table 2. Purificution nJ niitochondviul D N A po1~~merrr.w j?oni y u s i Yeast cells were grown for 14 h at maximal aeration in a medium containing 0.3'>. A. (1971) Riochwi. Biopti1,s. Rrs. f'onzinzrn. 44, 37 -43. 29. Springgate, C . F., Mildvan, A . S., Ahramson, R., Engle, J. L. & Locb, L. A. (1973) J . Biol. C'ht~iw. 248, 5987-5993. 30. Poiesz, B. J.. Seal, G. & Loeb, L. A . (1974) Pro(,. Ntrtl A m / . S(,i. C:.S.A. 71. 4892-4896. 31, Rama Reddy, G. V., Goulian, M. 81 Hendlci-. S. S . (1971) Nor. New Biol. 234, 286-288. 32. Momparler, R. L., Rossi, M. & Lahivan. A. (1973)J. Biol. Clrcni. 248,285-293. 33. Lynch, W. E. & Lieberman, J. (1973) Bioc,/rrni. Uiopl7j~.s.Rc.t. Corwnirn. 52, 843 - 849. -
REFERENCES 1. Borst, P. (1972) Annu. Rev. Bioclien?. 41, 333-376. _. 7 Kasamatsu, H. & Vinograd. J . (1974) Annu. Rea. Biochrm. 43, 695-719. 3. Wintersberger, E. (1974) Curr. Top. Microhiol. 6 6 , 77- 102. 4. Wintersberger, U. & Wintersberger, E. (1970) Eur. J . Biockt'fT7. 13. 20 - 27. 5. Kalf, F. G. & Ch'ih J. J. (1968) J . Biol. Chon. 243, 4904-4916. 6 . Mrqcr. R . R . B Simpson. M . V. (1970) .1. Bwl. ('hcvn. 245. 3426 - 3435. 7. 'ribbeits, C. J. 8. & Vinograd. J. (1973) J . Biol. Chcw?. 248, 3367 - 3379. 8. Fry, M. & Weissbach, A. (1973) Biocherni.s1r4; 12, 3602-3608. 9. Westergaard. 0. & Lindberg, B. (1972) Eur. J . Biochrnr. ZX, 422-431. 10. Wintersberger. U. & Wintersberger, E. (1970) Eur. J . Biod7er77. 13, 11-19. 11. Wintersbcrger, E. (1974) Eur. J . Biochern. 50, 41 -47. 12. Aposbian, H. V. & Kornberg, A. (1962) J . Biol. C h n . 237. 519 - 525. 13. Lowry. 0. M., Kosebrough, N . J., Farr, A. L. & Randall. R . J . (1951) .I. Bid. C h m . 193, 265-275.
U. Wintcrsberger and H. Blutsch, Institut f i r Krehsfoi-schung der Universitit Wien, Borschkegasse 8a. A-1090 Wien, Austria
