Mitochondrial DNA II. Sedimentation analysis and electron microscopy of mitochondrial DNA from chick liver

156

BIOCFIIMICA ET BIOPHYSICA ACTA

BBA 95711

MITOCHONDR1AL DNA II. S E D I M E N T A T I O N ANALYSIS AND E L E C T R O N MICROSCOPY OF MITOC H O N D R I A L DNA FROM C H I C K L I V E R

P. BORST*, E. F. J. VAN BRUGGEN**, G. J. C. M. RUTTENBERG*** AND A. M. KROON*

Department o/ l~Iedical Enzymology, Laboratory o[ Biochemistry, University o/ Amsterdam, Amsterdam (The Netherlands)*, Laboratory o/ Structural Chemistry, The University, Groningen (The Netherlands)**, and Laboratory o/Biochemistry and Toxicology, University o/A msterdam, Amsterdam (The Netherlands) *** (Received April I9th, 1967)

SUMMARY

I. The physicochemical properties of pure mitochondrial DNA from chick liver were studied b y band sedimentation in the analytical ultracentrifuge and b y electron microscopy. 2. Up to 8o % of native chick-liver mitochondrial DNA sedimented in a homogeneous band with an S2o,w = 39 S, the remainder of the high-molecular-weight DNA sedimenting in a homogeneous band with an S2o,w = 27 S. In some preparations a minor third component with an S2o,w = 24 S was present. The 39-S component was not affected b y the peptide hydrolase pronase, but it was converted into the 27-S component b y treatment with pancreatic deoxyribonuclease (EC 3.1.4.5) or hydroquinone, or b y "ageing". 3. Electron micrographs of all preparations of chick-liver mitochondrial DNA, spread according to the Kleinschmidt protein-monolayer technique, showed predominantly molecules in which no free ends could be distinguished. No branched molecules were seen in any preparation. 4. Micrographs of 39-S DNA, prepared b y preparative sucrose-gradient centri/ugation, contained 84 % highly twisted circles, 14 % open or half-open circles and I o/ /o linear molecules. Micrographs of pure 27-S DNA contained only 4 % twisted circles, 77 % half-open or open circular molecules and 19 % linear molecules. 5. The mean circumference of 63 more or less open molecules was 5-35 # with 9 ° % of all values falling between 4.85 and 5.85/z. 6. In mitochondrial DNA denatured in 12 % formaldehyde, 3 major and 3 minor components were found in analytical band-sedimentation studies using 3 M CsC1 containing 2 % formaldehyde as bulk solution. The major components were tentatively identified as the formaldehyde double-stranded cyclic coil (s~o,w = 83 S), Postal address: Jan Swammerdam Institute, Ie Constantijn Huygensstraat 2o, Amsterdam, The Netherlands. *

Biochim. Biophys. Acta, 149 (I967) 156-172

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the single-stranded ring (s~o,w ~ 32 S) and the single-stranded broken ring (S2o,w ---28 S). 7. In alkali, mitochondrial DNA sedimented as a heterogeneous collection of fragments. 8. We conclude that chick-liver mitochondrial DNA in situ is a doublestranded circular molecule with a molecular weight of the sodium salt of Io. lO811.1o 6. Both strands are covalently continuous and, on extraction, the DNA is obtained in a twisted circular form (S2o,w ---- 39 S) which is converted into an open circular form (s~0,w = 27 S) after cleavage of at least one phospho-diester bond. The 24-S component is tentatively identified as the linear form of mitochondrial DNA. 9. The close similarity of the physicochemical properties of mitochondrial DNA and the circular viral DNA molecules is stressed.

INTRODUCTION In the previous paper 1 we have described methods for the large-scale preparation of pure mitochondrial DNA from chick liver. In renaturation experiments 1-a this DNA behaved like a very homogeneous population of molecules with a molecular weight comparable to that of the DNA viruses. This suggested that the molecular weight of mitochondrial DNA might be low enough to permit its isolation intact even in the absence of special precautions to avoid shear degradation. Therefore an analysis of the molecular weight distribution of mitochondrial DNA from chick liver was undertaken. Sedimentation studies soon revealed the presence of two homogeneous components, sedimenting with S2o,w values of about 39 S (I) and 27 S (II), respectively 2. We showed by electron microscopy that both forms were circular ~-4 and we concluded a-5 that I represents the twisted circular form (c/. VINOGRADet al. 6) of mitochondrial DNA, in which both strands of the DNA are covalently continuous, while I I represents the open circular form in which at least one covalent bond is broken in at least one of the strands a-5. The electron micrographs and the sedimentation studies on which this conclusion was based are presented in detail in this paper.

MATERIALSAND METHODS

Chick-liver mitochondrial D N A This was isolated and purified as described in the previous paper 1.

Analytical band sedimentation Band sedimentation was carried out as described b y VINOGRADand co-workers 6-s in the Beckman-Spinco Model E analytical ultracentrifuge. A Kel-F bandforming centrepiece, type II, with 3 ° mm path length was used with 20-50/~1 of DNA (A260m~ z 0.5-4.0 per cm) in low salt and 1.7o ml of a bulk solution which Biochim. Biophys. Acta, 149 (i967) 156-I72

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consisted either of CsCI (0 = 1.35) in IO mM sodium phosphate buffer (pH 6.8), containing o.I or i.o mM sodium EDTA or of i M NaC1, 0.050 M sodium phosphate (pH 6.8), unless another solution is specified. Rotor speed was kept for 7 min at 5000 rev./min (c/. ref. 9) and then rapidly increased to 39 460 rev./min. Ultravioletabsorption photographs were taken on Kodak Professional film every 4 min after reaching full speed. Rotor temperature during the run was kept at 20 °. Tracings of photographs were made with a Beckman-Spinco Model R Analytrol densitometer. S2o,wvalues were calculated using the correction factors for solvent viscosity and density and DNA specific volume given by BRUNER AND VINOGRAD8 and STUDIER9.

Heat denaturation in the presence o~/ormaldehyde A sample of 20 #1 (A260m~ = 4.7 ° per cm) mitochondrial DNA was mixed with 20/,1 of 24 % formaldehyde freshly neutralized with NaOH in o.Io M sodium phosphate (pH 7.8), and heated for IO min in a sealed glass tube to the temperatures indicated. The DNA was then analysed by analytical band sedimentation using a CsC1 bulk solution of ~ = 1.35, containing io mM sodium phosphate (pH 6.8) and 2 ~o freshly neutralized formaldehyde.

Denaturation by alkali The DNA solution in either 1.2 M NaCl-0.o5 M sodium phosphate (pH 7.8) or in 7.5 mM sodium phosphate-o.I mM sodium EDTA (pH 7.0) was brought with cone. NaOH to a final NaOH concentration of o.I or 0.2 M and analysed by band sedimentation with either 0. 9 M NaC1 in o.I M NaOH or CsC1 (0 = 1.35) in 0.2 M NaOH as bulk solution.

Deoxyribonuclease treatmen! DNA samples were incubated for IO min at 25 ° in a mixture containing o.2 M NaC1, 0.03 M Tris-HC1 (pH 7.4), 4 mM MgCI~ and varying concentrations of pancreatic deoxyribonuclease (EC 3.1.4.5). The incubation was stopped by adding sodium EDTA (pH 8.0) to a final concentration of 0.04 M and the DNA was analysed on a bulk solution of CsC1 (0 = 1.35) containing IO mM sodium phosphate (pH 6.8) and I mM sodium EDTA.

Preparative sucrose-gradient centri/ugation A sample containing 50/~g of chick-liver mitochondrial DNA in I.I ml of 7.5 mM sodium phosphate (pH 7.o)-o.1 mM sodium EDTA was layered onto a linear gradient of sucrose from 2 1 % to 5 % (w/v) in o.15 M NaCl-Io mM sodium phosphate (pH 6.8)-o.1 mM sodium EDTA. The gradient was centrifuged in the SW 25 I rotor of a Beckman-Spinco Model 50 L preparative ultracentrifuge for 15 h at 2o ooo rev./min. After puncturing the bottom of the tube, fractions (14 drops) were collected.

Biochim. Biophys. Acta, 149 (1967) i56-i72

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159

Electron microscopy Specimens were prepared for electron microscopy with the spreading technique of KLEINSCHMIDTet al. 1°, with minor modifications. DNA solutions were dialysed against 1. 3 M ammonium acetate at 2 ° and diluted with this buffer to a final concentration of 2-4 #g/ml. Just before spreading, cytochrome e was added to o.oi °/o final concentration. A petri dish (diameter 20 cm) covered with a fresh sheet of Parafilm was used as a trough. This was filled with deionized water redistilled over quartz. The spreading of about o.I ml of the mixture of DNA and cytochrome c was initiated from a I cm × 4 cm piece of freshly cleaved mica wetted in advance with the trough solution. The spreading was followed by means of talcum powder. Grids covered with a carbon film were floated on the DNA-protein area for about 30 sec. After blotting with filter paper and drying in air they were shadowed for about IO sec under rotation (speed 30o0 rev./min) with 7 mg platinum at a distance of 5 cm and an angle of 8 °. Electron microscopy was carried out with a Philips EM-2oo at 60 kV using double-condensor lens illumination (focussed spot size about 5 /*) with 3oo/~ condensor-I, 200/~ condensor-II and 4 ° / , objective apertures. Use was made of the new type of electron gun with a 0.5 mm aperture in the wehneltcylinder, which gave a very coherent beam. The liquid-nitrogen-cooled anti-contamination device was routinely used. Pictures were taken at 7ooo-fold magnification on Ilford 5 B I I 35-mm film (developed 15 rain in Amaloco Glycinool) or at 20 ooo-fold on Ilford N4o plates (developed IO min in Kodak D76 ). The magnification was calibrated with a carbon grating made by Ladd with 216o lines/mm. Enlarged tracings (7- to I4-fold ) of the DNA molecules were traced at a total magnification of 49 000-98 ooo on paper by means of a Wild M5 stereomicroscope provided with a drawing tube. The length of the DNA molecules on the drawings was determined with a map measuring device.

Other analytical methods These were the same as described in the previous paper 1.

Materials Crystalline deoxyribonuclease I from bovine pancreas was obtained from Sigma, pronase from Calbiochem and cytochrome c from Boehringer. Parafilm was obtained from Marathon, Neenah, Wisconsin, and a 37 % formaldehyde solution (not containing methanol) was an Analar product from British Drug Houses. A sample of replicative-form DNA of phage # X 174 (see ref. I I ) was kindly donated b y Professor H. S. JANSZ. It contained more than 9 ° °/o component I, with an s20,w -- 21. 4 S (ref. 12).

RESULTS

Composition o//reshly prepared mitochondrial DNA Freshly prepared mitochondrial DNA from chick liver sediments in two comBiochim. Biophys. Acta, 149 (1967) I56--I7~

P. BORST et al.

16o

ponents with S2o,w values of 39 S (I) and 27 S (II), with no material sedimenting at intermediate S values. The sedimentation coefficients were determined in bandsedimentation experiments with I.O M NaC1 bulk solutions at low DNA concentrations; under these conditions the effect of DNA concentration on the sedimentation coefficient is negligible, according to STUDIER9. Similar sedimentation coefficients were obtained in earlier moving-boundary sedimentation experiments2, 4 in o.15 M NaCl-o.oI 5 M sodium citrate and in band-sedimentation experiments with CsC1 bulk solutions in which the sedimentation coefficient was calculated from the relative sedimentation velocities of mitochondrial DNA and the replicative-form DNA of q)X 174, added as an internal marker (Fig. I). Top

~eniscus

Bottom

L

Fig. i. Analytical band sedimentation of chick-liver mitochondrial DNA through neutral CsC1 bulk solutions. Conditions: see METHODS. Upper tracing: 4°/~1 13 387 chick-liver mitochondrial DNA (A260m# 2.78 per cm). Lower tracing: 2o/~1 B 387 chick-liver mitochondrial DNA with 2o~1 replicative-form DNA phage ~ X 174 (A260m# I.OO per cm). Pictures were taken 32 min (upper tracing) and 34 min (lower tracing) after reaching full speed. Sedimentation is from left to rigbt.

The relative amounts of I and II varied in 2o different preparations from 80 % I and 20 % II to IOO % II. Since I is easily converted into II during storage above o ° it seems likely that in situ the bulk of mitochondrial DNA is present as component I and that variable amounts of I are converted into II during the lengthy purification procedure. In many but not in all preparations of mitochondrial DNA some ultraviolet-absorbing material was present which did not sediment (c/. Figs. I, 4 and 8). In some cases this material was introduced during concentration of the DNA, in others it was present already in the DNA eluted from the methylated albumin on kieselguhr column. In the latter case it may represent DNA or RNA fragments not removed by tile column purification procedure.

Electron microscopy o/ I and I I Electron micrographs of freshly prepared chick-liver mitochondrial DNA contained predominantly molecules in which no free ends could be distinguished (Fig. 2). The molecules could be classified in four types: highly twisted molecules without free ends (Fig. 3a-c), half-open circular molecules (Fig. 3d-f), open circles (Fig. Biochim. Biophys. Acta, 149 (1967) 156-172

MITOCI-IONCRIALDNA. II

16I

Fig. 2. Electron m i c r o g r a p h of a r e p r e s e n t a t i v e field of chick-liver mitochondrial D N A at 49 ooo X magnification.

3g-i) and linear molecules. No branched molecules were seen in any of the preparations studied. For quantitation, both completely open molecules and those that were "untwisted" but that still retained a number of cross-overs (usually less than six), presumably as an artefact of preparation when the DNA attached to the grid film, were included in the category 'open circles'. Tile twisted molecules included those that had the twisted rod configuration, spread as illustrated in Fig. 3c, and those that were still closely wrapped as the rosette pattern in Fig. 3a. The number of crossovers in the twisted molecules could not be accurately counted, but it was always in excess of twenty. Circular molecules that could not be defined clearly as open or twisted, according to the criteria given above, were called half open (Fig. 3d-f). This category included those "open" molecules having an excessive number of crossovers (presumably as a result of how the DNA fell on the grid during preparation) and also those molecules having significant fractions of both open and twisted areas. To obtain preparations containing either I or I I the mitochondrial DNA was fractionated on a sucrose gradient, as shown in Fig. 4. The fractions indicated by the brackets were concentrated and dialysed to remove sucrose and analysed by band sedimentation in the analytical ultracentrifuge. The band-sedimentation experiments showed that the slow peak of the sucrose gradient contained only component n while the fast peak contained a mixture of about 80 % I and 2o % n . (The latter m a y well have been formed from I during concentration and dialysis.) Samples from fractions 16 and 22 of the sucrose gradient, indicated b y the arrows in Fig. 4 Biochim. Biophys. Acta, 149 (1967) 156-172

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Fig. 3. Different t y p e s of circular molecules p r e s e n t on electron micrographs of chick-liver mitoehondrial D N A (scale lines are o.2/~); a e, twisted molecules; d f, half-open circles; K-i, open circles.

Biochim. Biophys. Acta, i49 (I967) I56-172

MITOCI-IONDRIALDNA. II 0.6

I

I

I

I

163 I

Top

I

I

30

Bottom

04 ~~.M 0.5

27S

]-

39S

i

i t

i

25 20

~ 0.3 ¢J

15

~ O.2 c o

lO

b o.~ <

~3 E I

30

I

25

I

20 Froction

I

15 NO.

I

10

I

5

I

1

5

2 03.05

I-'I

I

4.05 Length

5.05 (p)

F i g . 4. Sucrose-gradient fractionation of chick-liver mitochondrial D N A see METHODS.

hTL 6.05

7.05

(B 3 6 2 ) . Conditions:

F i g . 5. Length distribution of circular D N A from chick liver.

and corresponding to I and II, respectively, were analysed by electron microscopy. The results of a semi-quantitative estimate of the distribution of the molecules over the four classes defined above are given in Table I. There is a close correlation between the presence of twisted molecules seen in electron micrographs and the presence of I in sedimentation studies. Component n seen in sedimentation studies can apparently give rise both to the half-open and open circular forms seen in electron micrographs. The linear molecules presumably represent circles broken during spreading. TABLE I DIFFERENT TYPES OF MOLECULES IN ELECTRON MICROGRAPI-IS OF PURIFIED 3 9 - S AND 27-S D N A FROM CHICK LIVER

T h e 3 9 - S D N A w a s a s a m p l e o f f r a c t i o n 16, t h e 2 7 - S D N A a s a m p l e o f f r a c t i o n 22 o f the sucrose gradient depicted in F i g . 4. T h e definition of the different t y p e s of molecules is given in the text.

Component 39 S

27 S

T w i s t e d circles ( % o f total) H a l f - o p e n c i r c l e s ( % o f total) Open circles ( % o f t o t a l ) Linear molecules ( % o f total)

84 3 ii

4 43 34

I

19

N u m b e r of molecules c o u n t e d

232

207

The contour length of 63 more or less open circular molecules was measured and the values found are given in Fig. 5. The mean contour length of chick-liver mitochondrial D N A was 5.35 # and 9 ° % of all values fell between 4.85 and 5.85/*. The circumference of the twisted circular molecules could not be accurately measured but their apparent length was compatible with a value of 5 #. Since HOTTA AND BASSEL1~ have reported that an occasional circular molecule Biochim. Biophys. Acta, 1 4 9 ( 1 9 6 7 ) 1 5 6 - 1 7 2

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of variable contour length was present in DNA extracted from boar sperm, nuclear DNA from chick liver purified and spread according to the same techniques used for mitochondrial DNA was also studied. No circular DNA at all was found after an intensive search. Conversion o~ I into I I Several treatments of mitochondrial DNA were found to lead to a loss of I with a concomitant increase in II. These treatments included prolonged storage at o-4 °, treatment with hydroquinone, rapid squirting through a No. 16 V2A-steel needle and incubation with pancreatic deoxyribonuclease. Treatment with pronase according to HOTTA AND BASSEL 13, however, had no effect. The results of an experiment with deoxyribonuclease are presented in Fig. 6. Resolution of the different components by band sedimentation was not optimal in this experiment because the bulk solution contained i mM EDTA, which led to a high background absorption. Nonetheless, it is clear that component I is converted into I I by deoxyribonuclease without formation of substantial ultraviolet-absorbing material of intermediate S values. A similar disappearance of I was found after incubation of mitochondrial DNA with hydroquinone as described by VINOGRAD et al. 6. Disappearance was not complete in these experiments even though the hydroquinone concentration used was five times as high as that used by VINOGRADgt al. 6. 27

39

¢1 0

P

0.14

o 0.70 o

14o r~

Direction of sedimentation

Direction of sedimentation

Fig. 6. A n a l y t i c a l b a n d s e d i m e n t a t i o n of chick-liver m i t o c h o n d r i a l D N A a f t e r t r e a t m e n t w i t h d i f f e r e n t c o n c e n t r a t i o n s of p a n c r e a t i c d e o x y r i b o n u c l e a s e , as indicated. Conditions: see .~ETHODS. T h e figure h a s b e e n a s s e m b l e d f r o m p h o t o g r a p h s t a k e n 32 m i n a f t e r r e a c h i n g full speed in 4 d i f f e r e n t r u n s . T h e h i g h b a c k g r o u n d a b s o r p t i o n of t h e CsC1 b u l k solution is d u e to t h e presence of I m M E D T A . Fig. 7- A n a l y t i c a l b a n d s e d i m e n t a t i o n of chick-liver m i t o c h o n d r i a l D N A a f t e r p r o l o n g e d dialysis a g a i n s t o.i m M s o d i u m E D T A (pH 7.o). (a) F r e s h l y p r e p a r e d chick-liver m i t o c h o n d r i a l D N A ( E x p t . 335) ; 5o-/*1 s a m p l e (A260m/~ = 1.2 per cm) in 7.5 m M s o d i u m p h o s p h a t e (pH 7.o) c o n t a i n i n g o.i m M s o d i u m E D T A , a n a l y s e d on i.o M N a C l - o . o 5 M s o d i u m p h o s p h a t e (pH 7.o) b u l k solution; p h o t o g r a p h t a k e n 24 rain a f t e r r e a c h i n g 39 460 r e v . / m i n . (b) As a; p h o t o g r a p h t a k e n 32 m i n a f t e r r e a c h i n g 39 460 r e v . / m i n . (c) Chick-liver m i t o c h o n d r i a l D N A ( E x p t . 335) after c o n c e n t r a t i o n a n d p r o l o n g e d dialysis a g a i n s t o.i m M s o d i u m E D T A (pH 7.o); 4o-/~1 s a m p l e (A260m# = 6.3 per cm) a n a l y s e d on CsC1, e = 1.35 b u l k solution; p h o t o g r a p h t a k e n 24 rain a f t e r r e a c h i n g 44 77 ° r e v . / m i n . (d) As e; p h o t o g r a p h t a k e n 32 m i n a f t e r r e a c h i n g 44 77 ° r e v . / m i n . T h e arrows indicate t h e 24-S c o m p o n e n t in F r a m e s c a n d d.

Biochim. Biophys. Acta, 149 (1967) 156-172

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165

In some of our recent preparations of mitochondrial DNA a minor third component was just visible sedimenting immediately behind component I I with an s20,w value of 24 S. This component, designated component I I I , was more pronounced in one preparation which had been dialysed for a long time against o.I mM sodium E D T A (pH 7.0). As shown in Fig. 7 all component I had disappeared in this preparation after dialysis and it appears as if component I I I was formed from I or II.

Men~seus

20 °

I*II

40"

I+X

50 °

60 °

I÷X

70 °

I*T

80 o

I*X

90 °

I*X

100 °

I÷X

80 °

80 °

I+X

90 °

I+~

> Direction

of sedimentotion

Fig. 8. A n a l v t i c a l b a n d s e d i m e n t a t i o n of m i t o c h o n d r i a l D N A a f t e r h e a t i n g in t h e p r e s e n c e of f o r m a l d e h y d e . M i t o c h o n d r i a l D N A (Expt. B 26) w a s h e a t e d for io m i d a t t h e t e m p e r a t u r e ind i c a t e d as d e s c r i b e d u n d e r METHODS a n d a n a l y s e d b y a n a l y t i c a l b a n d s e d i m e n t a t i o n t h r o u g h a CsC1 b u l k s o l u t i o n c o n t a i n i n g 2 % f o r m a l d e h y d e . T h e figure h a s b e e n a s s e m b l e d f r o m p h o t o g r a p h s t a k e n a b o u t 20 miD a f t e r r e a c h i n g full s p e e d w i t h t h e e x c e p t i o n of t h e l a s t t w o f r a m e s w h i c h were t a k e n 4 ° m i d a f t e r r e a c h i n g full speed. T w o p r e p a r a t i o n s were a n a l y s e d , one f r e s h l y isolated c o n t a i n i n g b o t h c o m p o n e n t I a n d II, t h e o t h e r c o n s i s t i n g o n l y of c o m p o n e n t II, purified b y prep a r a t i v e s u c r o s e - g r a d i e n t c e n t r i f u g a t i o n , as s h o w n in Fig. 4. T h e p h o t o g r a p h s were arbitrarily matched at the meniscus.

Biochim. Biophys. Acta, 149 (1967) 156-172

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P.

BORSTet al.

Sedimentation analysis o~ denatured mitochondrial DNA When tile intact circular DNA of any of several viruses is denatured by alkali it is converted into a component with a very high sedimentation coefficient, designated the double-stranded cyclic coilla, 14. Many attempts to identify a similar form of mitochondrial DNA in alkali were unsuccessful. The alkali-denatured DNA sedimented as a broad heterogeneous band with a maximal sedimentation coefficient of 20-25 S. With some preparations there was no material sedimenting with S values above IO, even when more than 50 °~o of the native DNA consisted of I and when special care was taken to avoid shear degradation. To exclude artefacts introduced by the denaturing conditions a preparation of mitochondrial DNA containing 75 % I and 25 °~o U was mixed with replicative-form DNA of phage OX 174 and the mixture was denatured by alkali and analysed on alkaline CsC1 by band sedimentation. Although the DNA of ~bX 174 was converted into its 53-S double-stranded cyclic coil (see ref. i2), the mitochondrial DNA again sedimented in a broad heterogeneous band with a maximal S value of 25 S. In further studies the DNA was therefore denatured by heat in the presence of 12 % formaldehyde and analysed on CsC1 bulk solutions containing 2 % formaldehyde as described by CRAWrORDTM. The results of these experiments are illustrated in Figs. 8 and 9. Heating mitochondrial DNA in the presence of formaldehyde to 60 ° or higher leads to the formation of three major components. The two slow components with calculated s20,w values of about 28 S and 32 S, respectively, sediment as one band in Frames 4-7 and 9 of Fig. 8 but they can be distinguished as separate bands after longer centrifugation times as shown in the lower 2 frames of Fig. 8. The two slow components are also found in the denatured, purified component II, but the fast component is only formed in DNA containing both I and U. The sedimentaNATIVE 90

I

I

DENATURED

I

80 7C

83S

so

1,39S

I

o

o 5o

5 ~4e

0 0 o

0

E

0 o

2O

o

O 0

o O

0 0

I

I

I

i

20 ~

40 °

60 °

80 °

0 0

m,2~s

i 100

°

Heating temperature

i1,275 "'---"

28S

Fig. 9. S e d i m e n t a t i o n coefficients of m i t o c h o n d r i a l D N A a f t e r h e a t i n g to d i f f e r e n t t e m p e r a t u r e s in t h e p r e s e n c e of f o r m a l d e h y d e . C o n d i t i o n s : see METHODS a n d t h e l e g e n d of Fig. 8. O, m a j o r bands; O, minor bands. Fig. IO. D i a g r a m m a t i c r e p r e s e n t a t i o n of t h e v a r i o u s f o r m s of m i t o c h o n d r i a l DNA, m o d i f i e d f r o m a p a p e r b y VINOGRAD et al. s on p o l y o m a v i r a l DNA. The d a s h e d circles a r o u n d t h e d e n a t u r e d forms indicate the relative h y d r o d y n a m i c diameters. Biochim. Biophys. Acta, 149 (i967) 156-172

MITOCHONDRIAL

DNA. I I

167

tion coefficient of the fast component rises as a function of temperature to a m a x i m u m at 80 °. The relative amount of all three maior components decreases with increasing temperature especially above 80 ° and none of them is found in DNA heated at ioo ° for IO min. While the amounts of 28-S and 32-S material are about equal in DNA heated at 80 ° or lower, there is definitely less 32-S than 28-S material at 9 °° both in the total mitochondrial DNA and in the purified component I I DNA. After heating at 5 °0 we observed in two experiments that the major component sedimented more rapidly than the minor component as if a reversal in the sedimentation coefficients of I and I I had occurred. An interpretation for this phenomenon is given in the discussion. In addition to the major components three minor components were observed, one of which is very pronounced in DNA heated at 9 °0 (c]. Fig. 8). The presence of these components was not influenced by varying the concentration of formaldehyde in the bulk CsC1 solution between o and 12 %. The component sedimenting with a sedimentation coefficient of about 35 S was the only minor component present after heating purified component II, the others being only observed in preparations containing both I and II. The amount of the 44-S component was variable in different experiments with the same DNA heated at 80 ° and it m a y well be due to DNA crosslinked b y the formaldehyde treatment. The nature of tile other minor components is under study.

DISCUSSION

The physicochemical properties of DNA from chick-liver mitochondria are very similar to those observed b y others for the circular DNA's of several DNA viruses, as shown in Table I I and III, and our results smoothly fit the scheme proposed by VINOGRADet al2 to interpret the various DNA species observed in the sedimentation analysis of polyoma viral DNA, as illustrated in Fig. IO. According to this interpretation components I and I I observed in freshly prepared mitochondrial DNA in neutral concentrated salt solutions represent the twisted circular and open TABLE

II

SEDIMENTATION COEFFICIENTS OF THE VARIOUS FORMS OF NATIVE CIRCULAR D N A , ISOLATED FROM DIFFERENT SOURCES All s e d i m e n t a t i o n c o e f f i c i e n t s w e r e d e t e r m i n e d in n e u t r a l h i g h - s a l t s o l u t i o n ; I , I I a n d I I 1 r e f e r t o the twisted circular, the open circular and the linear forms, respectively.

Source o / D N A

Length

(~) P o l y o m a v i r u s 14 1.58 q ) X 174 r e p l i c a t i v e form12,17 1.64 H u m a n p a p i l l o m a virus1% 18 2. 7 Chick mitochondria* 5.4 L a m b d a v i r u s 19 ~1 17

S,o,to values I

II

III

I/II

II /III

2o. 3 22.o 28.2 39 48.~

15.8 17.o 20.2 27 36-*

14. 4

1.3 1.3 1. 4 1.4 1. 3

i. i o

18.o 24 32

1.12 i. i o 1.14

" This paper. *~ R e c a l c u l a t e d f r o m t h e d a t a i n r e f . 20.

Biochim. B i o p h y s . . 4 c t a , I 4 9 ~I967) I 5 6 - I 7 2

P. BORST el al.

168 TABLE III SEDIMENTATION FROM

DIFFERENT

COEFFICIENTS

OF T H E V A R I O U S D E N A T U R E D

FORMS OF CIRCULAR

DNA,

ISOLATED

SOURCES

I I , native circular D N A in n e u t r a l high salt; Ia, double-stranded cyclic coil in alkali; It, doubles t r a n d e d cyclic coil in CsC] containing formaldehyde; SSR and SSB, single-stranded intact and b r o k e n circle respectively in alkali or in CsC1 containing formaldehyde.

Source o / D N A

P o l y o m a virus s q~X 174 replicative form 1= H u m a n papilloma virus TM Chick mitochondria***

s2~,w values I1

I~

If

SSR

SSB

Ia/II

If/H

15.8 I7-o 20.2 27

53 53

41.

18-4 18.4

15.7 14.3

3.4 3.

2.6

32

28

65 ** 83

3. 2 3.1

* Recalculated f r o m d a t a in ref. 22. ** Recalculated from d a t a in ref. 16. *** This paper.

circuaar forms respectively of mitochondrial DNA, while component I I I which appears in "aged" preparations under similar conditions represents the linear form of mitochondrial DNA. In I both strands of the circle are covalently continuous. The higher sedimentation coefficient of I than of I I is explained not by a difference in mass but b y a difference in hydrodynamic volume as indicated by the twisted structure in Fig. IO. VINOGRAD AND LEBOWITZ15 have recently suggested that the folded structure of I arises because the pitch of the DNA helix in the cell is larger than the pitch of the helix in the Watson-Crick structure in the purified DNA. The change in pitch during isolation leads to "tertiary turns" in the circle if both strands are continuous. Scission of at least one covalent bond in at least one of the strands of I, for instance b y an endonuclease, introduces a swivel and the structure can unfold to form the open circle II. If both strands are cut at the same site a linear molecule is formed. On denaturation, the helix of I cannot unwind when the hydrogen bonds are broken and a very compact "supercoil" structure is formed in which all turns of the Watson-Crick helix are conserved. On the other hand II can unwind on denaturation and will yield single-stranded circles, single-stranded complete linear molecules, and single-stranded fragments depending on the amount of preexisting single-strand breaks in II. The following evidence supports this interpretation in the case of mitochondrial DNA: Freshly prepared mitochondrial DNA analysed in neutral concentrated salt solution contains I and I I in variable proportions as the only sedimenting ultraviolet-absorbing molecular species. Purified II, free of ultraviolet-absorbing contaminants, is circular as shown by electron microscopy (c/. Table I). The average contour length of the open circles is 5.4/*, which is about the value expected for a circular DNA with an s20,w = 2 7 S (see below). I is converted into II (without formation of material of intermediate sedimentation coefficients) by the same treatments which convert the twisted circular forms of certain DNA viruses into the corresponding open circular forms. These treatments probably all lead to the scission of covalent bonds. A decrease in sedimentation Biochim. Biophys. Acta, 149 (i967) 156 i72

MITOCHONDRIAL

DNA. I I

169

coefficient by a factor o. 7 corresponds either to a substantial increase in hydrodynamic volume or to a decrease in molecular weight b y a factor 2.6 according to STUDIER'S9 formula for linear DNA's. The latter alternative is excluded by two observations. First, 80 °/o of the molecules present in electron micrographs of I had a twisted knot-like appearance* without detectable free ends, like the twisted circular forms of other DNA's (see refs. 14, 18, 23, 24). Although accurate length measurements of these molecules were not possible, it is clear from some of the examples presented in Fig. 3 that their length is close to that of the open circular molecules. Secondly, the fact that the sedimentation coefficient of I after heating in the presence of formaldehyde increased with increasing temperature up to 80 °, while that of II reached a constant value already at 6o °, would be difficult to understand if the difference between I and II was a difference in molecular weight, On the other hand this phenomenon is readily explained b y the known resistance to denaturation of a circular DNA which cannot unwind because both strands are covalently continuous. In the case of polyoma DNA this resistance leads to a difference in melting point between components I and II of more than 20 ° (see ref. 15). The melting point of pure component I of mitochondrial DNA has not yet been determined. The identification of the other components depicted in Fig. io is only tentative. Component I I I was found in a preparation in which all I originally present had disappeared after dialysis against o.I mM EDTA. The sedimentation coefficient of this compound is that expected, on the basis of STUDIER'S formula 9, for the linear form of mitochondrial DNA in which the circle is opened without lowering the molecular weight. Definite identification of this compound will require a demonstration of its formation from I or I I b y a deoxyribonuclease which cuts both strands of the DNA simultaneously, followed b y a verification of its linear nature by electron microscopy (c/. ref. 6). The single-stranded circular and linear derivatives of the mitochondrial DNA were tentatively identified as such on the basis of three observations: (i) they are formed from I I on denaturation; (ii) the sedimentation coefficients are those expected from the work of others with viral DNA (Table I I I ) for the single-stranded circular and linear forms of a molecule with a molecular weight of about 5.2" IOe daltons; (iii) the amounts of the 32-S and the 28-S forms are about equal after heating at 80 °, but after heating at 9 °0 tile amounts of both are diminished and the decrease in the 32-S component is more marked than that of the 28-S component, This is exactly what would be expected for the random thermal degradation of a mixture of circles and linear molecules in which the scission of one phospho-diester bond converts the circle into the linear molecule, while scission of the linear molecule leads to a decrease of the population. Although the results obtained thus far with mitochondrial DNA are very similar to those obtained earlier for the circular DNA of DNA viruses there are a few minor differences. In o.I M NaOH component I was not converted into a rapidly sedimenting alkaline "supercoil" form as observed with the DNA viruses (c/. Table III) but it sedimented as a variable and heterogeneous collection of fragments. Similar * As p o i n t e d o u t b y VINOGRAD AND LEBOWlTZ1~ the highly twisted f o r m of I found in elect r o n m i c r o g r a p h s does n o t necessarily r e p r e s e n t the f o r m of I p r e s e n t in solution. The latter is called t w i s t e d only to indicate its c o m p a c t structure.

Biochim. Biophys. Acta, 149 (1967) I 5 6 - I 7 z

i7o

v. BORST et al.

results have been obtained with component I isolated from rat-liver mitochondria, using either chloroform or phenol for deproteinization*. It is unlikely that this is due to inadequate analytical procedures since conversion of I into the formaldehyde "supercoil" could be easily demonstrated, while, moreover, under our conditions component I of the replicative-form DNA of phage qbX 174 was converted into the corresponding alkaline supercoil form. It is possible that the isolated mitochondrial DNA contains depurinations (see refs. 14 and 25), either preexistent or introduced during purification. This point is under investigation. A second point requiring further investigation is the result obtained after heating mitochondrial DNA in the presence of formaldehyde at 5 o°. In two experiments we observed that the more rapidly sedimenting band contained more ultraviolet-absorbing material than the slower sedimenting band in contrast to the situation observed after heating at 40 ° or 60 ° . We interpret this phenomenon, which has not been observed for the viral DNA's, as follows: Slight denaturation of I will allow the unwinding of the tertiary twists and the sedimentation coefficient of I will therefore drop and approximate that of II, as shown for component I of several viruses16, 22. More extensive denaturation of I will lead to the gradual formation of the formaldehyde supercoil with the attendant increase in sedimentation coefficient. Denaturation of I I will at first lead to an increase in sedimentation coefficient until a temperature is reached where the two strands separate when the sedimentation coefficient should abruptly drop to that of the mixture of single-stranded rings and linear molecules16, 22. Since I is more resistant to denaturation than I I (see ref. 15) it is to be expected, however, that at any temperature between the start of denaturation and complete denaturation, I will be less denatured than II. At intermediate temperatures, therefore, nearly completely denatured I I could sediment more rapidly than slightly denatured I. No explanation can be offered for the minor components consistently found in sedimentation experiments after denaturation with formaldehyde. A similar minor component was observed by CRAWFORD16 under similar conditions with papilloma DNA; its nature was not identified. We now turn to the question of the structure and molecular weight of mitochondrial DNA in situ. In the previous papers1,2, 4 it was shown that a large proportion of the DNA present in chick-liver mitochondrial preparations can be obtained as high-molecular-weight DNA, banding at 1.7o8 g/cm 8 in CsC1, while we have shown in this paper that in some preparations up to 80 ~o of this high-molecularweight DNA is present as I, the remainder sedimenting as II. These results show that the bulk of mitochondrial DNA is present as circular DNA and that in nearly all of these circles the two strands are covalently continuous. The results obtained after denaturation of mitochondrial DNA in the presence of formaldehyde indicate that the molecules which sediment as component I I in neutral salt contain one strand which is covalently continuous. Therefore, none of the circles found in purified mitochondrial DNA could have been formed b y the circularization of linear molecules with complementary terminal single-stranded regions 21 during lysis of the mitochondria. Although the available evidence (reviewed in ref. 4) indicates that mitochon* E. M. SMIT, G. J. C. M. RUTTENBERG AND P. BORST, u n p u b l i s h e d o b s e r v a t i o n s .

Biochim. Biophys. Acta, 149 (1967) 156-172

MITOCHONDRIALDNA. I I

I71

drial DNA replicates in the organelle, branched molecules were absent in all our preparations of chick-liver mitochondrial DNA, as we have pointed out previously a and in a series of preparations of mitochondrial DNA from ox, sheep, mouse, rat and duck tissues s. This might mean that mitochondrial DNA does not replicate in the mitochondrion, or that replicating molecules are eliminated b y the purification and spreading procedures employed, or that the proportion of replicating molecules is so small that the chance of finding one is very small. The latter possibility is not unreasonable in view of the report 2e that the half-life of mitochondrial DNA in rat liver is about 9 days. If mitochondrial DNA replicated as slowly as the nuclear DNA of cells in tissue culture -- about 0.5 #/rain (see refs. 27 and 28) -- only I in iooo molecules would be branched. However, if the replication rate is similar to that of bacterial DNA this proportion would be near i to IOO ooo and in this case one cannot expect to detect branched molecules in the absence of special enriching procedures. From our results the molecular weight of the sodium salt of intact mitochondrial DNA from chick liver can be calculated by three methods. Using a value of 1.92. lO6 daltons DNA per # (ref. I9), the average contour length of 5.35 # is equivalent to a molecular weight of lO.3" lO6. Secondly, the molecular weight can be calculated by comparing the contour length of mitochondrial DNA with that of the replicative-form DNA of phage ¢~X 174 spread and photographed under exactly the same conditions. For the latter an average contour length of 1.7o/~ is obtained ~, which is in good agreement with the 1.64 /~ reported by KLEINSCHMIDT et alJ v. SINSHEIMER29 has found a molecular weight of 1. 7" 1os for single-stranded ~bX 174 DNA in light-scattering experiments. Therefore, the molecular weight of mitochondrial DNA is (5.35/1.7o) • 1. 7 • lO6. 2 = lO.7" lO6. Thirdly, the molecular weight of mitochondrial DNA can be calculated by making use of STUDIER'S9 equation for the relation between sedimentation coefficient and molecular weight of linear DNA's and assuming that the linear form of mitochondrial DNA has a sedimentation coefficient of 24 S (c/. Table III). The value found is lO. 9- lO6. The agreement between the molecular weights found b y three different methods is good and we conclude that the molecular weight of the sodium salt of chick-liver mitochondrial DNA is IO. IO6-Ii • lOs. The consequences of this low molecular weight for the suggested genetic function of this DNA have been discussed in a previous paper 4. The limited data available for mitochondria from other organisms already indicate that circularity of mitochondrial DNA may be a general phenomenon. Circular molecules with a contour length of about 5.4 # have been found in mitochondrial DNA from duck s, mouse2-4,3°, 31, rat5, 3°**, ox 2-4, sheep 5, carp** and fly** tissues. The bulk of mitochondrial DNA from duck, rat and sheep tissues 5 sedimented with the same sedimentation coefficients as components I and II of chick mitochondrial DNA. These results are compatible with the idea that also in duck, rat and sheep mitochondria component I is the predominant species of mitochondrial DNA present in situ. The finding that mitochondrial DNA from such varied sources has about the same size is rather intriguing. Possible explanations for this phenomenon will be considered elsewhere. * E. F. J. VAN ~c~RUGGEN AND H. S. JANSZ, u n p u b l i s b e d observations. ** E. F. J. VAN BRUGGEN, M. M. C. LEURS, P. BORST, A. M. KROON AND G. J. C. M. R.UTTENBERG, u n p u b l i s h e d results.

Biochim. Biophys. Acta, 149 (1967) 156-172

172

P. BORST et al.

AC KNOWLEDGEMERrTS

We are grateful to Professor E. C. SLATER and Protessor M. GRUBER for helpful suggestions and encouragement; to Mr. E. M. SMIT for his collaboration in some oi these experiments; to Professor H. S. JANSZ and Mr. L. VAN I~ESTEREN (University of Leiden) for advice and generous help with ultracentrifuge equipment; to Miss C. M. RUNNER, Miss E. BAAN and Mr. H. D. BATINK for expert technical assistance; and to Mr. S. NOORMAN for his skillful photography. REFERENCES I P. BORST, G. J. c. M. RUTTENBERG AND A.M. KROON, Biochim. Biophys. Acta, 149 (1967) 14o. 2 P. BORST AND G. J. C. M. RUTTENBERG, Biochim. Biophys. Acta, 114 (1966) 645. 3 E. F. J. VAN BRUGGEN, P. BORST, G. J. C. M. RUTTENBERG, M. GRUBER AND A. M. KROON, Biochim. Biophys. Acta, 119 (1966) 437. 4 P. BORST, A. M. KROON AND G. J. C. M. RUTTENBERG, in D. SHUGAR, Genetic Elements: Properties and Function, Academic Press and P. W. N., L o n d o n and W a r s a w , 1967, p. 81. 5 A. M. KROON, P. BORST, E. F. J. VAN BRUGGEN AND G. J. c. M. RUTTENBERG, Prec. Natl. Acad. Sci. U.S., 56 (1966) 1836. 6 J. VINOGRAD, J. LEBOWlTZ, R. RADLOFF, R. V~rATSON AND P. LAIPIS, Prec. Natl. Acad. Sci. U.S., 53 (1965) 11o4. 7 J. VINOGRAD, R. BRUNNER, R. KENT AND J.WEIGLE, Proc. Natl. Acad. Sci. U.S., 49 (1963) 902. 8 R. BRUNER AND J. VINOGRAD, Biochim. Biophys. Acta, lO8 (1965) 18. 9 F. W. STUDIER, J. Mol. Biol., i i (1965) 373. io A. I~. KLEINSCHMIDT, D. LANG, I). JACHERTS AND R. K. ZAHN, Biochim. Biophys. Acta, 61 (I962) 857. i i H. S. JANSZ, P. H. I~OUWELS AND J. SCHIPHORST, Biochim. Biophys. Acta, 123 (1966) 626. 12 P. H. POUWELS, H. S. JANSZ, J. VAN ROTTERDAM AND J. A. COHEN, Biochim. Biophys. Acta, 119 (1966) 289. 13 Y. HOTTA AND A. BASSEL, Prec. Natl. Acad. Sci. U.S., 53 (1965) 356. 14 R. WEIL, J. VINOGRAD AND W. STOECKENIUS, Prec. Natl. Acad. SeN. U.S., 5 ° (1963) 73 ° 15 J. VINOGRAD AND J. LEBOWlTZ, J. Gen. Physiol., 49 (1966) lO3. 16 L. V. CRAWFORD, J. Mol. Biol., 13 (1965) 362. 17 A. K. KLEINSCHMIDT, A. BURTON AND R. L. SINSHEIMER, Science, 142 (1963) 961. 18 L. V. CRAXVFORD, E. M. CRAWFORD, J. P. RICHARDSON AND H. S. SLAYTER, J. Mol. Biol., 14 (1965) 593. 19 L. A. 3/ICHATTIE AND C. A. THOMAS, Jr., Science, 144 (1964) 1142. 20 V. C. ]3ODE AND A. D. KAISER, J. Mol. Biol., 14 (1965) 399. 21 A. D. HERSHEY, E. BURGI AND L. INGRAHAM, Proc, Natl. Acad. Sci. U.S., 49 (1963) 748. 22 L. V. CRA~vVFORDAND P. H. BLACK, Virology, 24 (1963) 388. 23 D. S. RAY, A. PREUSS AND P. H. HOFSCHNEIDER, J. Mol. Biol., 21 (1966) 485 . 24 R. JAENISCH, P. H. I-IoFSCHNEIDER AND A. PREUSS, J. Mol. Biol., 21 (1966) 5Ol. 25 ~V. FIERS AND R. L. SINSHE1MER, J. Mol. Biol., 5 (1962) 420. 26 D. NEUBERT, Arch. Exptl. Pathol. Pharmakol., 253 (1966) 152. 27 J. CAIRNS, J. Mol. Biol., 15 (1966) 372. 28 J. H. TAYLOR, personal c o m m u n i c a t i o n . 29 R. L. SINSHEIMER, J. Mol. Biol., I (1959) 43. 3 ° J. H. SINCLAIR AND ]3. J. STEVENS, Prec. Natl. Acad. Sci. U.S., 56 (1966) 508. 31 M. M. K. NASS, Prec. Natl. Acad. Sci. U.S., 56 (1966) 1215.

Biochim. Biophys. Acta, 149 (1967) 156-172