Mitochondrial DNA

BIOCHIMICA ET BIOPHYSICA ACTA

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M I T O C H O N D R I A L DNA V. A 25-/* CLOSED C I R C U L A R D U P L E X DNA MOLECULE IN W I L D - T Y P E Y E A S T M I T O C H O N D R I A . S T R U C T U R E AND G E N E T I C C O M P L E X I T Y

C. P. H O L L E N B E R G * , P. B O R S T * AND E. F.

j.

VAN BRUGGEN**

"Department o/ Medical Enzymology, Laboratory o] Biochemistry, University o[ Amsterdam and **Laboratory o[ Structural Chemistry, The University, Groningen (The Netherlands) (Received D e c e m b e r I l t h , 1969)

SUMMARY

I. Electron micrographs of DNA released b y osmotic shock from isolated mitochondria of Saccharomyces carlsbergensis NCTC 74 and S. cerevisiae H I contained closed circular duplex DNA molecules. 2. The mean length of 6 closed circular molecules from S. carlsbergensis mitochondria was 25.6:~o. 9/* and the mean length of fourteen molecules from S. cerevisiae mitochondria was 25.o-t-l.O/*. The number of cross-overs for both yeast species was 6.5 per/* length. The majority of the linear molecules was longer than 15/*, with lengths up to 27/*. The DNA released from the mitochondria of S. cerevisiae H I also contained three smaller closed circular molecules (3, 4 and IO/~). 3. All attempts to isolate the 25-/* circles intact were unsuccessful. DNA isolated from yeast mitochondria b y a direct lysis method followed b y CsC1 equilibrium gradient centrifugation consisted exclusively of linear molecules of 2-24/* with a modal length at 7-8/*. 4. A closed circular DNA fraction of nuclear density was present in the CsC1 equilibrium gradients of yeast lysates in the amount of 1- 4 °/o of the nuclear DNA. The majority of these circles had an average contour length of 2/*, but circles up to 7/* were present. 5. The renaturation of mitochondrial DNA of S. carlsbergensis and S. cerevisiae followed second-order kinetics with a renaturation constant about I . I - I . 6 times that of T4 DNA, determined under identical conditions. 6. We conclude that the intact mitochondrial DNA of S. carlsbergensis and S. cerevisiae consists of a circular molecule with a contour length of approx. 25/* and a genetic complexity equivalent to this size.

INTRODUCTION

The size and structure of yeast mitochondrial DNA have been the subject ot controversial reports. In previous work in this laboratory 1,~ mitochondrial DNA from " Postal address: J a n S w a m m e r d a m I n s t i t u t e , le Constantijn H u y g e n s s t r a a t 2o, A m s t e r d a m , The Netherlands.

Biochim. Biophys. Acta, 209 (197 o) 1 - I 5

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C . P . HOLLENBERG et al.

Saccharomyces carlsbergensis was found to consist of heterogeneous linear molecules up to 18 #. We concluded that these were fragments of a larger molecule, fragmented b y the procedures used for its release from the mitochondria. Subsequent reports from other laboratories presented results that seemed not compatible with this conclusion. AVERS et al. 3 reported that up to 50 % of the DNA molecules released from yeast mitochondria by osmotic shock were circular, the contour length varying between I and IO/~, without preferred size classes. Circular molecules were also observed by GUI~RINEAUet al. 4 and SHAPIRO et al. 5. These authors noted, in addition, that the linear molecules in their DNA preparations were distributed in preferred length classes 5,6. According to SHAPIRO et al. 5, part of the linear molecules appeared to have complementary single-stranded 'sticky' ends. This paper presents a reinvestigation of the size and structure of yeast mitochondrial DNA, initiated in an a t t e m p t to resolve the discrepancies in the results obtained in different laboratories. Our results strongly indicate that the intact mitochondrial DNA of wild-type yeast is a closed circular duplex with a contour length of about 25/* and a genetic complexity commensurate with this size. The main results of this work have been reported in preliminary form 7.

MATERIALS AND METHODS

Yeast species and culture conditions S. carlsbergensis NCTC 74 and S. cerevisiae H I , haploid, adenine-requiring mutant derived from S. cerevisiae, Hansen, CBS 5493. Cells were grown aerobically at 28 ° in complete medium containing per 1:5 g pepton, 2.5 g yeast extract, 2 g KH2PO 4, 6 g (NH4)2SO 4, 0.5 g M g S Q ' 7 H~O, 0. 5 g CAC12.6 H20, 5 g glucose and 4 g sodium lactate or in minimal medimn according to GALZY AND SLONIMSKI ~, supplemented with 0.5 % glucose, 0. 4 O//osodium lactate and 3.6 mg E3Hladenine or E14Cladenine per 1 (final specific activity resp. 1. 5 or o.12 mC/1). Cells were harvested in tile late log-phase if grown in complete medium and in the mid log-phase if grown in the minireal medium. Preparation o[ cell lysates /or CsCl equilibrium gradients Cells were converted into spheroplasts according to the method of DUELL et al. 9 as modified by KovA~ et al. 1°. Spheroplasts were washed twice with o.3 M mannitol0. 7 M sorbitol-2 mM E D T A (pH 7.6). The spheroplast pellet was suspended in icecold water, immediately followed b y addition of I vol. of ice-cold o.i M Tris-HC1, 20 mM EDTA, 15 mM NaCI-I.5 mM sodium citrate buffer (pH 7.o), 4 °/o Sarkosyl (final pH 8.5). After gentle mixing CsC1 was added to a density of 1. 7 or 1.55 g/ml. In the latter case ethidium bromide to a final concentration of 2-4 mg/ml was also added. Preparative equilibrium centriJugation and analysis o] gradients ]or radioactivity Lysates were centrifuged in the angle 5o-rotor in a Spinco Model L2 centrifuge at 44 ooo rev./min for 42 h at 22 °. The tubes were punctured with a hollow needle and fractions dripped. The fractions were incubated in 0.5 M NaOH for I6 h at 37 °. After addition of z o o # g bovine serum albumin and sodium pyrophosphate (pH 6 Biochim. Biophys. Acta, 209 (197 o) 1-15

STRUCTURE OF MITOCHONDRIAL D N A

3

to a final concentration of 6o raM), the DNA was precipitated with trichloroacetic acid (final concn. 5 %)- The precipitate was collected on Whatman glass-fibre filters and washed 5 times with 3 ml 5 % trichloroacetic acid, containing 60 mM sodium pyrophosphate. After drying in counting vials and addition of IO ml toluene, containing 5 g 2,5-diphenyloxazole and 62.5 mg 1,4-bis-(5-phenyloxazolyl-2)-benzene per 1, radioactivity was counted in a Nuclear Chicago liquid scintillation counter. In general only one in two fractions was counted. The other fractions were stored for further analysis. Analytical ultracentrifugation was carried out in a Spinco Model E centrifuge, as previously described 11. Preparative band sedimentation through neutral CsC1 bulk solutions was carried out by the procedure of TOMIZAWAAND ANRAKU1=in the SW-5 o rotor in a Spinco Model L-I centrifuge.

Preparation o/yeast mitochondrial DNA /or electron microscopy Yeast mitochondria were prepared according to a modification of the method of K o v / ~ et al. 1°. Yeast spheroplasts were suspended in 0.35 M sucrose-o.I °/o bovine serum albumin-I mM EDTA (pH 7.6) and homogenized for 20 sec in a precooled Braun mixer. The homogenate was centrifuged for IO rain at 15oo ×g. The supernatant was centrifuged io min at 8000 xg. The pellet was suspended in o.35 M sucrose containing 5 mM MgC12 and 200/~g pancreatic deoxyribonuclease (EC 3.1.4.5) per ml. After 2o rain at o ° the suspension was centrifuged at 12oo x g for IO min. The mitochondria were pelleted from the supernatant by centrifugation at 17 ooo ×g for IO rain and suspended in 0.35 M sucrose-o.I M EDTA (pH 8.o) followed by repelleting at 17 ooo ×g for IO rain. A fluffy layer covering the mitochondrial pellet was washed off by 0.35 M sucrose-o.I M EDT& (pH 8.0). For osmotic lysis the mitochondria were diluted in 4 M ammonium acetate, kept for 15 rain at o ° and after mixing with cytochrome c spread on icecold water. After IO rain the monolayer was picked up with grids covered with a carbon film and treated further as described by VAN BRUGGEN et al}. Electron microscopy of purified DNA was carried out with the protein monolayer technique as previously described 2.

Large-scale isolation o/ yeast mitochondrial DNA Mitochondria treated with deoxyribonuclease as described above were suspended in I vol. of 0.35 M sucrose-o.I M EDTA (pH 8.0). For lysis I vol. of a solution containing 4 % Sarkosyl, 8 % 4-aminosalicylate, 2 % tri-isopropylnaphthalene sulphonate and 6 % 2-butanol was added, followed by 2 vol. phenol, supplemented with m-cresol and 8-hydroxyquinoline according to KIRBYla. After shaking for 30 rain at room teulperature the phases were separated by centrifugation and 2 vol. of ethanol were added to the water phase. After one night at --20 ° the precipitate was pelleted, dissolved in 15 mM NaCI-I.5 mM sodium citrate buffer (pH 7.0) and adjusted with CsC1 to a density of 1. 7 g/ml. The solution was centrifuged for 42 h at 44 ooo rev./min in the angle 50 rotor in a Spinco Model L-I centrifuge. The tubes were fractionated by drop collection from the bottom in fractions of about 0.07 ml. A possibly present minor nuclear DN A band in the gradients of these large-scale preparations was detected by measuring the fluorescence of the fractions after addition of 0. 7 ml of a dilute ethidium bromide solution (IO/,g/ml). Mitochondrial DNA fractions were Biochim. Biophys. Acta, 209 (197 o) 1-15

4

¢. P. HOLLENBERGet al.

pooled, dialyzed for 18 h against 15o mM NaCI-I5 mM sodium citrate buffer (pH 7.0) and incubated with a mixture of pancreatic (20#g/ml) and T, ribonuclease (o.o15 #g/ml) for 3° rain at 3°0 (pancreatic ribonuclease was heated for IO rain at at 80 ° before the ineubationlX). The DNA was further purified by chromatography on a one-layer column of methylated albumin on kieselguhr as previously described 11. By dialysis the DNA was brought in the appropriate salt concentration. If required, the DNA solution was concentrated under reduced pressure at about 3o°. Renaturation o] D N A DNA used for renaturation studies was exhaustively dialyzed against 15 mM NaCI-I.5 mM sodium citrate buffer (pH 7.0) and 2-ml portions were sonicated for 5 periods of I rain in a MSE Ioo-W sonic disintegrator at maximal output. During sonication the solution was kept at o 4 ° and continuously flushed with either helium or nitrogen gas. This led to fragments with a single-strand molecular weight of 2.5" lO5, determined by alkaline band sedimentation (see below). DNA renaturation was studied by the following procedure: o.I 0.5 ml DNA solution in 15 mM NaC1 1.5 mM sodium citrate buffer (pH 7.0) was heated for Io rain in a quartz cuvette, sealed with a Teflon stopper at 920 in a water bath. The cuvette was then dried and transferred to the cuvette chamber of a Gilford 2400 spectrophotometer, maintained at renaturation temperature (25 ° below the melting temperature of the DNA in 15o mM NaC1 15 mM sodium citrate buffer (pH 7.0). After 4 rain concentrated preheated NaCl-sodium citrate solution was rapidly added to a final volume of I.O ml and a final concentration of 15o mM NaC1 15 mM sodium citrate buffer (pH 7.0). The A260nm was recorded as a function of time after mixing. At the end of the experiment the temperature of the cuvette chamber was raised to 95 ° and the Ae60 ..... of the completely denatured DNA was recorded. Renaturation rates are expressed as the second-order renaturation constant k2, calculated with the formula of WETMUR AND DAVIDSON J4, slightly modified to allow corrections for the hypochromicity of single-stranded DNA at the renaturation temperature and for contaminants in the DNA solution that absorb light of 260 nm. Other D N A preparations DNA's from guinea-pig liver mitoehondria 11, bacteriophage T 4 (refs. 15 and 16), bacteriophage 7t (refs. 16 and I7), Escherichia coli (ref. 18) and ~2P-labelled replicatire form DNA of bacteriophage ~ X I 7 4 (ref. 19) were obtained by standard procedures. 3H-labelled chick-liver mitochondrial DNA was prepared by the procedure of BORST et al. ix from mitochondria incubated with E~H]dATP in vitro ~°. Fragment size o / D N A The fragment size of sonicated DNA was determined by band sedimentation through alkaline CsC1, as described by VINOGRADand co-workers21,2e, using a MSE analytical ultracentrifuge. 30-50 #1 DNA in 15 mM NaCI-I.5 mM sodium citrate buffer (pH 7.0) were centrifuged through 1.7o ml of a bulk solution of CsC1 (p = 1.35 g/ml) in o.I M NaOH at 44 ooo rev./min. The sedimentation coefficients in 3 M CsC1 were converted to s20,~, in I M NaC1, using linear DNA of bacteriophage ~ X I 7 4 as standard. The S2o,~ values in I M NaC1 were used to calculate the single-strand fragment size with the formula of STUDIER23. Biochim. Biophys. ,4cta, 209 (197o) 1--1,5

STRUCTURE OF MITOCHONDRIAL O N A

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RESULTS

Analysis o/yeast DNA /or closed circular duplex molecules by equilibrium centri/ugation o/cell lysates in CsCl, containing ethidium bromide The presence of closed circular DNA molecules in yeast DNA was studied by CsC1 adding to a spheroplast lysate and centrifuging the mixture to equilibrium with and without ethidium bromide. Spheroplasts were used rather than purified mitochondria to minimize the chance of degradation of mitochondrial DNA. To raise the detection level for DNA in the gradients an adenine-requiring m u t a n t grown on radioactive adenine was used in these experiments. Representative gradients with and without ethidium bromide are shown in Figs. i a and Ib. In all experiments a large fraction

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Fig. I. a. CsC1 g r a d i e n t fractionation of whole-cell D N A from a s p h e r o p l a s t lysate of S. cerevisiae H I , g r o w n in m i n i m a l m e d i u m containing [14C]adenine. F r a c t i o n v o l u m e o.125 ml. See MATERIALS AND METHODS for f u r t h e r e x p e r i m e n t a l details, b. Same as a b u t in the presence of e t h i d i u m bromide (EB). F r a c t i o n v o l u m e 0.08 ml. c. F r a c t i o n s of B a n d C in b recentrifuged in a CsC1 g r a d i e n t w i t h o u t e t h i d i u m bromide. E t h i d i u m b r o m i d e was r e m o v e d b y c h r o m a t o g r a p h y on a Dowex-5o column. As m a r k e r ( - - - ) , 3H-labelled nuclear D N A (p = 1.7Ol-}:o.oo2 g/ml) was added. Fraction v o l u m e 0.04 ml. Only t h e p a r t of the g r a d i e n t containing r a d i o a c t i v i t y is shown.

of the acid-insoluble, alkali-stable radioactivity was present in the protein cake at the top of the gradient (not shown in Fig. I). This cake probably consists mainly of incompletely lysed spheroplasts, because there was no selective trapping of either mitochondrial or nuclear DNA. In the absence of ethidium bromide the remainder of the acid-insoluble, alkali-stable radioactivity was recovered in two bands, the nuclear DNA band at 1.7o14-o.oo2 g/ml and the mitochondrial DNA band at 1.6844-o.oo2 g/ mh These values, which were determined b y analytical CsC1 gradient centrifugation in separate experiments, are very similar to those obtained with other strains of Biochim. Biophys. Acta, 2o9 (197 o) 1-15

c.P. HOLLENBERGet al.

6

Saccharomyces (c/. ref. 24, Table II). In gradients containing ethidium bromide 1-40J/o of the acid-insoluble, alkali-stable radioactivity was present in a third band C at a density about 25 mg/ml higher than the nuclear DNA band (Fig. Ib). At this position in the gradient dosed circular duplex DNA m a y be expected to band. The DNA present in this Band C was further characterized in two ways: (I) After removal of ethidium bromide the DNA was centrifuged to equilibrium in CsC1 in the presence of 14C-labelled nuclear DNA. The result of this experiment presented in Fig. IC, showed that Band C DNA had an equilibrium density 1-2 mg/ml higher than that of the bulk of nuclear DNA. This indicates that Band C consists of closed circular duplex DNA derived from the nucleus, since mitochondria only contain D N ~ with an equilibrium density of 1.684 g/ml (ref. I and see below). (2) Electron micrographs of the gradient fractions of Band C contained highly twisted DNA molecules without free ends, with some open circular molecules. The contour lengths of twenty-six molecules that could be traced are presented in Fig. 2. The majority of the molecules

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were about 2 # long, but longer molecules were also present. The length distribution is similar to that previously observed for open circles of nuclear density by SINCLAIR et al. ~°. In none of the gradients with ethidium bromide analyzed, a band was detected at a position expected for closed circular mitoehondrial DNA. To raise the limit of detection the region of the gradient indicated by the arrow was scanned for circular molecules b y electron microscopy. None were found, To exclude the unlikely possibility that mitochondrial circles were hidden in the main nuclear DNA peak, fractions from the heavy side of this band were recentrifuged in CsC1 without ethidium bromide and in the presence of a marker DNA in the same way as in the experiment shown in Fig. IC. No DNA of mitochondrial density was detected. These results suggest that closed circular duplex DNA with a contour length smaller than 5 # does not represent a major fraction of the mitochondrial DNA of our yeast strain.

Characterization ol isolated yeast mitochondrial DNA by band sedimentation and electron microscopy Spheroplast lysates were centrifuged to equilibrium in CsC1 containing ethidium bromide and fractions corresponding to the main nuclear and the mitochondrial peaks were analyzed by band sedimentation through neutral CsC1 and by electron Biochim. Biophys. Acta, 209 (197 o) 1-I 5

S T R U C T U R E OF M I T O C H O N D R I A L D N A

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F i g . 3. P r e p a r a t i v e b a n d s e d i m e n t a t i o n of i s o l a t e d a l l - l a b e l l e d y e a s t m i t o c h o n d r i a l a n d n u c l e a r D N A c a r r i e d o u t b y t h e p r o c e d u r e of TOMIZAWA AND ANRAKU TM. O.I m l of t h e D N A s o l u t i o n w a s l a y e r e d o n 3 m l 3 M CsC1 c o n t a i n i n g o . o i M T r i s b u f f e r ( p H 7.1) a n d o v e r l a y e r e d w i t h i m l l i q u i d p a r a f f i n . T u b e s w e r e c e n t r i f u g e d i n t h e S W - 5 o r o t o r i n a S p i n c o M o d e l L - I c e n t r i f u g e for 3 h a t 3 ° ooo r e v . / m i n a t 12 °. G r a d i e n t s w e r e f r a c t i o n a t e d a n d a n a l y z e d a s d e s c r i b e d u n d e r MATERIALS AND METHODS. F r a c t i o n v o l u m e o.I m l . A s m a r k e r 3 H - l a b e l l e d c h i c k - l i v e r m i t o c h o n d r i a l D N A c o n t a i n i n g C o m p o n e n t I (39 S), I I (27 S) a n d s o m e d e g r a d e d D N A w a s s e d i m e n t e d i n a separate tube.

microscopy. The results of these experiments are presented in Figs. 3 and 4. The bulk of the mitochondrial D N A sedimented in a broad band with a mean sedimentation coefficient similar to that of the open circular form of chick mitochondrial D N A (s20,w -- 27 S) used as marker in these experiments. The sedimentation profile does not show the multiple size classes, observed by others. The bulk of the nuclear D N A sedimented even faster than 39 S. Electron micrographs of D N A from the mitochondrial peak of a CsC1 gradient containing ethidium bromide contained only linear molecules varying in length bePURIFIED LINEAR

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F i g . 4- H i s t o g r a m of l e n g t h s of i s o l a t e d y e a s t m i t o c h o n d r i a l D N A of S. cerevisiae H I . T h e D N A w a s d e r i v e d f r o m t h e p o o l e d f r a c t i o n s of a m i t o c h o n d r i a l b a n d in a CsCl g r a d i e n t .

Biochim. Biophys. Acta, 209 (197 o) 1 - 1 5

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Fig. 5. E l e c t r o n m i c r o g r a p h s of t w i s t e d c i r c u l a r D N A m o l e c u l e s r e l e a s e d f r o m y e a s t m i t o c h o n d r i a , l y s e d b y o s m o t i c s h o c k a s d e s c r i b e d u n d e r MATERIALS AND ~iErltODS. T h e s c a l e line is o. 5/~. A - C . S. cerevisiae, l e n g t h r e s p e c t i v e l y : 26, 28 a n d 25 i~. D. S. cerevisiae i s o - N , l e n g t h 24/z.

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STRUCTURE OF MITOCHONDRIAL D N A

9

tween 2 and 24 F with a mode a t 7-8 # (see Fig. 4). Not one circular molecule was seen in the mitochondrial DNA fractions from CsCI gradients of spheroplast lysates of either S. cerevisiae or S. carlsbergensis centrifuged to equilibrium in CsC1 in the absence of ethidium bromide.

Electron microscopy o / D N A released/rom isolated mitochondria by osmotic shock Tile results presented in the previous sections suggest (el. ref. 2) that the intact DNA of yeast mitochondria is at least 20 # long and that this DNA breaks in fragments during its isolation. In an a t t e m p t to visualize these postulated large molecules, yeast mitochondria were lysed by osmotic shock. In this procedure the DNA released is directly fixed in the protein monolayer and the chance of accidental degradation b y shear or nucleases is minimal. Before lysis all mitochondria] preparations were incubated with a high concentration of pancreatic deoxyribonuclease (see MATERIALS AND METHODS) to remove any contaminating nuclear DNA present (c/. ref. 2). Five mitochondrial preparations were analyzed: two from S. cerevisiae, three from S. carlsbergensis. Under the electron microscope all preparations yielded mainly large networks of long DNA strands enmeshed in mitochondrial debris. All preparations contained in addition areas of DNA with the typical twisted appearance of closed circular duplex DNA (e/. ref. 2). In four separate preparations twisted circles were found sufficiently free of overlying molecules or mitochondrial debris to allow unambiguous contour-length measurements. Four of these are shown in Fig. 5 and another one in Fig. I of ref. 7- A histogram of all circles found is presented in Fig. 6. S.carlsbergensis 8

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Fig. 6. L e n g t h d i s t r i b u t i o n of t w i s t e d circles released f r o m y e a s t mitochondria, lysed b y osmotic shock as described u n d e r MATERIALS AND METHODS. Mitoehondria were isolated from S. carlsbergensis NCTC 74 a n d S. cerevisiae H I .

The striking result is that the majority of the circles fall in a homogeneous size class with an average contour length of 25 #. The appearance of the molecules is very similar to that of the closed, duplex circles released from animal mitochondria and this is confirmed by a quantitative analysis of cross-overs (c/. ref. 2). On the average the 25-/* circles from yeast mitochondria contained 6.5 cross-overs per # whereas 6. 7 cross-overs per # were found in the twisted circular DNA released from chick-liver mitochondria. Biochim. Biophys. dcta, 209 /I97 o) 1-15

IO

c.P. HOLLENBERGet al.

In addition to the closed circles one open circle was found with contour length of 27 ~. The linear molecules present that could be unambiguously traced were mostly longer than Io # but no molecules longer than 27 # were observed. Additional attempts to obtain small circular D N A [rom yeast mitochondria

Although the results presented in the previous sections are consistent in themselves and in agreement with previous work in this laboratory, they are incompatible with reports from other laboratories that up to 50 % of all yeast mitochondrial DNA m a y be present in the form of (predominantly open) circles varying in length between I and IO/~. A series of additional experiments was, therefore, done, all designed to retrieve the elusive small circles. Since all these experiments were negative we shall only brie[ly summarize the main approaches tried. To see if closed circular duplex DNA was degraded during the CsC1 equilibrium gradients, replicative-form of DNA bacteriophage 9 X I 7 4 was added immediately after lysing the spheroplasts and the mixture was centrifuged to equilibrium in CsCIcontaining ethidium. More than 75 % of the added radioactivity was recovered in one peak in the gradient at the position of closed circular duplex DNA. The following variations were tried in the isolation of spheroplasts and DNA: (i) Spheroplasts were prepared by the method of DUELL et al. 9, with the modification used b y AVERS26 and the DNA was isolated according to AVERS26. (2) To release membrane-bound DNA, spheroplast lysates were incubated with pronase (3 mg/ml) for 4 h at 3 0° before centrifugation to equilibrium in CsC1. (3) Spheroplasts were lysed i~, the presence of a large excess of calf-thynms DNA or yeast RNA to saturate nucleases present. (4) Different conditions for culturing the yeast cells were tried. In none of these experiments was a closed circular duplex peak found in CsCl-ethidium gradients at the position expected for mitoehondrial closed circular duplex DNA, although a nuclear closed circle band was always present, showing that the nuclear circles can be extracted under a variety of conditions. In a number of experiments fractions of the mitochondrial DNA peak in the CsC1 gradient were analyzed b y electron microscopy. In all cases linear DNA heterogeneous in length (as in Fig. 4) was observed and not a single circle was found. SHAPIRO el al. 5 have reported that their preparations of mitochondrial DNA contained a substantial fraction of linear molecules with single-stranded, complementary, 'cohesive' ends. By annealing these could be converted into molecules sedimenting slightly faster in sucrose gradients. We annealed purified mitochondrial DNA from S. cerevisiae by the procedure of RITCHIE et al. 27 and analyzed the preparation b y electron microscopy. No circles were found. Since small circles might be present in some Saccharomyces strains but not in others, Dr. Avers kindly sent us the wild-type S. cerevisiae strain Iso-N used in the experiments of AvERs et al. ~. The mitoehondrial DNA of this strain was studied by electron microscopy in mitoehondrial preparations lysed b y osmotic shock. The general appearance of these preparations under the electron microscope was very similar to that of the other strains studied. Several twisted circles were found in the 25-/* range but only one of these (shown in Fig. 5d) was free of overlapping molecules or debris. In addition two open circles of 4 and 6/, and two twisted circles of 6 # were observed. No circles smaller than 2 ~, were found. The fact that four good circles smaller than 25/J were found in this preparatiol~ against only one good 25-l~ Biochim. Biophys. Acta, 209 (I97o) 1-15

S T R U C T U R E OF M I T O C H O N D R I A L D N A

II

circle could indicate that small circles are more frequent in the Avers' strain than in the other strains studied, in which only three small circles against twenty large ones were found. Nevertheless, even in Avers' strain much less than I °/o of the total DNA released from the mitochondria was present as small circles in our experiments against up to 50 % in the experiments of AVERS et al. ~. Moreover, whereas preparations made in Avers' laboratory contained sufficient numbers of linear filaments smaller than IO # to construct meaningful histograms, such molecules were virtually absent in our preparations. This observation and the fact that we find 25-/~ circles in these preparations suggests that our inability to isolate small circles in quantity is not due to degradation of mitochondrial DNA.

Analysis o] the genetic complexity o/yeast mitochondrial DNA by quantative renaturation studies Under standard conditions the rate at which a denatured DNA renatures is inversely proportional to its complexity 14,~s, defined as the total number of base pairs in non-repeating sequences. By comparing the renaturation rate of an unknown DNA with that of a number of standard DNA's of known complexity, the complexity of the unknown can be calculated. We have used this technique to determine whether the genetic complexity of yeast mitochondrial DNA is commensurate with its genome size. Mitochondrial DNA from yeast was prepared on a large scale as described in MATERIALSAND METHODS. Both preparations used in the renaturation analysis were free of contaminating nuclear DNA, as shown by the results of the analytical CsC1 equilibrium gradient presented in Fig. 7. The DNA was sonicated to fragments of t I

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g

k ~ 1.7OlII II ~4 1.71o1.731 Density (glml I

F i g . 7. D e n s i t o m e t e r t r a c i n g of a n a n a l y t i c a l C s C l - d e n s i t y g r a d i e n t of m i t o c h o n d r i a l D N A isol a t e d o n a l a r g e - s c a l e f r o m S. carlsbergensis m i t o c h o n d r i a a s d e s c r i b e d u n d e r MATERIALS AND METHODS. T h e g r a d i e n t c o n t a i n e d I / z g Micrococcus lysodeikticus D N A (p = 1.731 g / m l ) a n d 2 / ~ g of E. coli D N A (p = 1.71o g / m l ) a s m a r k e r .

approx. 75o nucleotides and renaturation was followed in 15o mM NaCI-I 5 mM sodium citrate buffer (pH 7.o), as described under MATERIALSAND METHODS. Fig. 8 shows that renaturation followed simple second-order kinetics for at least 4 ° °/o of the renaturation reaction. There is no indication for the presence of components renaturing more rapidly than the bulk of the DNA. In Table I the average renaturation constants of the yeast mitochondrial DNA's are compared with the constants obtained for reference DNA's of known genome size fragmented to the same fragment size and renatured under the same conditions as yeast mitochondrial DNA. Biochim. Biophys. Acta, 209 ( I 9 7 o) 1 - 1 5

I2

C . P . HOLLENBERG et a~,

a

b

S. c a r l s b c r g e n s i s

S. c e r e v i s i a ¢

20

20

(6.~)

o

,

O

10

,

,

,

,

,

30 TIME (rain)

,

o

60

,

O

10

,

,

,

30 TIME (rnin)

60

Fig. 8. S e c o n d - o r d e r r a t e p l o t of t h e r e n a t u r a t i o n of t w o y e a s t m i t o c h o n d r i a l p r e p a r a t i o n s . T h e d a t a are p r e s e n t e d a c c o r d i n g to WETMUR AND DAVIDSON 14. T h e v a l u e s b e t w e e n b r a c k e t s are t h e s e c o n d - o r d e r r e n a t u r a t i o n c o n s t a n t s , c a l c u l a t e d from t h e slope of t h e line b y a p r o c e d u r e s l i g h t l y m o d i f i e d fro m \VETMUR AND DAVIDSON la. T h e a r r o w s i n d i c a t e t h e p o i n t a t w h i c h 5 ° % r e n a t u r a t i o n w a s r e a c h e d . F o r f u r t h e r e x p e r i n l e n t a l d e t a i l s see MATERIALS AND METHODS. a. S. carlsbergensis NCTC 74. b. S. cerevisiae H I .

The interpretation of these data is complicated by the facts that the renaturation constants of the reference DNA's are not exactly proportional to genome size. This is probably due to an effect of base composition (c/. ref. I4). Since no reference DNA's are available with a G + C content as low as yeast mitochondrial D N A (17 % G + C ; G. BERNARDI, personal communication), an accurate estimate of its genetic complexity is not possible but it is clear from the data in Table I that--considering its low G + C c o n t e n t - - t h e renaturation rate of mitochondrial D N A is fully compatible with the size of its genome.

TABLE

[

RENATURATION GENOME

RATES

SIZE AND BASE

OF

YEAST

MITOCHONDRIAL

DNA

AND

REFERENCE

DNA's

RELATED

TO

COMPOSITION

R e n a t u r a t i o n c o n s t a n t s were d e t e r m i n e d as d e s c r i b e d u n d e r MATERIALS AND METHODS. E v e r y v a l u e for t h e reference D N A ' s is t h e a v e r a g e of a t l e a s t t h r e e d e t e r m i n a t i o n s . The c o n s t a n t s for y e a s t m i t o c h o n d r i a l D N A r e p r e s e n t t h e e x t r e m e v a l u e s from a t l e a s t five e x p e r i m e n t s . The molec u l a r w e i g h t a n d base c o m p o s i t i o n of t h e reference D N A ' s were o b t a i n e d from t h e fol l ow i ng sources: E. coli a n d p h a g e T~ DNA, WETMUR AND DAVIDSONI~; p h a g e 2 D N A , THOMAS AND MACIrIATTIE~9; g u i n e a - p i g m i t o c h o n d r i a l DNA, BORST AND KROON 24. The ba s e c o m p o s i t i o n of y e a s t m i t o c h o n d r i a l D N A w a s o b t a i n e d from G. BERNARDI (personal c o m m u n i c a t i o n ) .

Source o[ D N A

E. coli P h a g e ), Guinea-pig mitochondria Phage T 4 S. carlsbergensis S. cerevisiae

Renaturation constant (1.mole 1.sec-1)

:Ilol, wt. genome (×±o 7)

(.)

(b)

0.8 54 I oo 6 7.1 9-9 6.4-9. 4

Biochim. Biophys. Acta, 209 (197 ° ) I 15

250 3.3 i, i 13 5 5

a ×b ( × zo ~)

G-a- C (mole 0/o)

2.0 1.8

5° 49

i. i 0.8 0.4 o.5 o.3 0.5

42 34 17 ~7

S T R U C T U R E OF M I T O C H O N D R I A L

DNA

13

DISCUSSION

To the variety of DNA molecules extracted in different laboratories from yeast mitochondria this paper adds a new class of twisted 25-# circles. The following arguments support the idea that these circles represent intact yeast mitochondrial DNA: twenty-one were found in the yeast strains analyzed, one S. carlsbergensis and two S. cerevisiae strains. Smaller circles were present in much lower frequency. Since the chances of finding an intact, free circle should decrease with the contour length, this observation rules out that the 25-/~ circles are multimers of a smaller monomer. Although many grids had to be scanned to collect a total of twenty-one circles, this does not mean that these circles were very rare in these preparations. Yeast initochondria do not lyse as completely as animal mitochondria and it was, therefore, difficult to find 25-/* circles completely free of debris and overlapping molecules. Hence, for every circle included in Fig. 6, at least ten other circles were discarded because they could not be unambiguously traced. Even when this is taken into account, linear DNA was much more frequent in all our preparations than 25-# circles. The linear molecules were very heterogeneous in size, however, and no molecules longer than 27 # were observed. It seems reasonable, therefore, to assume tlmt these linear molecules were derived from the 25-/* circles. It is highly unlikely that the 25-/* circles could be derived from non-mitochondrial contaminants present in the mitochondrial preparations. Nuclear DNA was removed by preincubating the mitochondria with deoxyribonuclease prior to osmotic shock. The effectiveness of this treatment is shown by the absence in our preparations of 2-/~ circles, the major class of nuclear circles (c/. Fig. 2). Other organelles susceptible to osmotic shock, impermeable to deoxyribonuclease and containing DNA are not known in yeast. Furthermore no viruses are known that infect yeast and it would be rather a coincidence if we would have found the first yeast virus simultaneously in 3 different healthy yeast strains. Taken together, these arguments strongly suggest that the 25-/z circles are the intact monomers of the yeast mitochondrial genome. 25 # is equivalent to a molecular weight (sodium salt) of 49' lO6. Since contour length measurements on twisted circles tend to be low, this value must be considered a minimum estimate. All attempts to isolate intact 25-# circles free of protein have failed up till now. Although linear DNA up to 24 # long was obtained no open 25-/~ circles were present. Even in the preparations lysed by osmotic shock only one open 27-# circle was found and even this one was not perfect. Three explanations could be considered for these results. (I) Nearly all mitochondrial DNA molecules in yeast spheroplasts are engaged in replication or recombination, and replicating molecules fragment during isolation. This explanation implies that intact 25-# circles are more easily found in DNA released from mitochondria by osmotic shock than in purified mitochondrial DNA. This is very unlikely. (2) Yeast contains active nucleases like spleen acid deoxyribonuclease that break both strands of a double-stranded DNA molecule at the same site. These nueleases are activated during lysis and they effect the fragmentation of all mitoehondrial DNA in the few seconds before they are inactivated by the detergent-CsC1 mixture. In the very dilute suspensions in 4 M ammonium acetate, used in the osmotic shock experiments, these nucleases might be unable to degrade all mitochondrial DNA molecules released. (3) Mitochondrial DNA is attached to a mitochondrial membrane and this attachment can only be severed at the cost of a Biochim. B i o p h y s . Acta, 2 0 9 (197 ° ) I - I 5

14

c.P. HOLLENBERGgt al.

double-strand break. We cannot decide between possibilities (2) and (3) with the information at hand. In addition to the 25-# circles, a few smaller circles were observed in electron micrographs of shocked mitochondria. These were typical twisted circles that could be unambiguously traced and it is, therefore, impossible that they are artifacts of the isolation or spreading procedures. Assuming that the chance of finding a circle is inversely related to its length, it is likely that the small circles are overrepresented in Fig. 6 and that these circles form only a small minority of the total mitochondrial DNA in wild-type yeast cells. A possible explanation for their presence is provided be preliminary observations with mitochondrial DNA of cytoplasmic petite mutants. We already reported 7 that the mitochondrial DNA isolated from a petite m u t a n t approximately eighty generations after induction b y ethidium bromide consisted nearly exclusively of circles smaller than I/~. It was possible to isolate these molecules intact by centrifugation of the whole lysate in a CsC1 gradient. Apparently, the mutation not only changed the size and density of the DNA, but also its sensitivity to degradation. The decrease in degradation could be the result of the decrease in circle size or possibly it might have to do with the change in membrane properties caused directly or indirectly by the petite mutations. On the basis of these results with a cytoplasmic petite mutant, the presence of small circles in mitochondrial DNA preparations of wild-type could be attributed to defective DNA extracted from the 1 % spontaneous petite ceils, always present in yeast cultures, and possibly from petite mitochondria present in normal cells. We have previously raised the possibility that the level of defective DNA in wild-type yeast cultures varies from strain to strain and that differences between strains could be responsible for the widely different percentage of small circles found in yeast mitochondrial DNA in different laboratories. The fact that the strain that yielded up to 5o ~o circular DNA in Avers' laboratory released hardly any small circles in this laboratory argues against this interpretation, unless small differences in growth conditions could be decisive. Although we cannot account for this discrepancy in results two points m a y be stressed: In the work reported from other laboratories nearly all small circles were of the open type. Whereas closed circular duplexes cannot arise as a consequence of isolation artifacts, it is more difficult to exclude this possibility for open circles. Secondly, it should be noted that the large majority of the linear DNA molecules obtained from yeast mitochondria in this laboratory were longer than 7 u, whereas most of the small circles found by others were smaller than 7/*- It is difficult to envisage how small circles could give rise to long linear molecules during the extraction of the DNA. Thirdly, the absence of circular molecules in preparations of DNA after denaturation and renaturation 3° makes the presence of a high percentage of small circles in the mitochondria very unlikely. These arguments suggest that the small circles are fragments of the mitochondrial genome produced by mutagenic events or as a consequence of the extraction and isolation of the DNA (open circles). The quantitative renaturation studies presented in this paper show that yeast mitochondrial DNA renatures about at the rate expected on the basis of its genome size. No evidence for repeating sequences was found. This imphes that, notwithstanding its exceedingly low G + C content of 17 mole °/o, the 75 ooo base pairs of the mitochondrial genome of yeast are present in a unique sequence without major reBiochim. Biophys. Acta, 209 (197o) 1-15

STRUCTURE OF MITOCHONDRIAL

DNA

15

dundancy. Potentially, the genetic information content of yeast mitochondria is, therefore, 5-fold higher than that of animal mitochondria. ACKNOWLEDGEMENTS

We are grateful to Professor E. C. Slater and Dr. A. M. Kroon for advice and to Miss W. Van Der Vegte and Miss H. M. Moerdijk for expert technical assistance. We are indebted to Dr. G. J. C. M. Ruttenberg and Mr. H. D. Batink for the analytical CsC1 gradient equilibrium centrifugation; to Mr. E. M. Smit for the analytical alkaline band sedimentation runs; to Mr. R. W. J. Thuring for carrying out most of the renaturation experiments; to Mr. L. Reijnders for the qJXI74 replicative form, [32P]DNA; to Mr. J. Ter Schegget for the chick-liver mitochondrial [~H]DNA; to Dr. J. C. Avers for sending yeast strains and to Dr. G. Woolfe of Boots Pure Drug, Nottingham, Great Britain, for a gift of ethidium bromide. This work was supported (in part) by The Netherlands Foundation for Chemical Research (S.O.N.) with financial aid from the Netherlands Organization for the Advancement of Pure Research

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Biochim. B i o p h y s . Acta, 209 (I97O) I - I 5