Chromosoma (Berl.) 82, 583-593 (1981) CHROMOSOMA �9 Springer-Verlag 198l

A DNA Binding Protein from Xenopus laevis Oocyte Mitoehondria

Monique Barat and Bernard Mignot te Laboratoire de Biologie G6n6rale, Bfitiment 400, Universit~ Paris-Sud, 91405 Orsay, France

Abstract. A D N A binding protein has been isolated, by affinity chromatogra - phy on D N A cellulose, f rom mi tochondr ia and f rom purified m i t D N A - protein complexes f rom oocytes ofXenopus laevis. This 12,500 daltons protein is polymeric in its native form and binds to D N A with a high efficiency. It exhibits an apparent ly preferential binding to the single-stranded fiber of the D loop structures.

Introduction

It is now beyond doubt that mitochondria l D N A (mi tDNA) is not " n a k e d " in the organelle but is associated with proteins (Kuroiwa, 1976; Pinon et al., 1978; Van Tuyle et al., 1979; Olszewka et al., 1980; R ickwood et al., submitted). M i t D N A is thus condensed and acquires a chromatin-l ike condi t ion as has been observed in Xenopus laevis (Pinon et al., 1978) and Paramecium aurelia (Olszewka et al., 1980). A basic protein has also been purified f rom yeast mito- chondr ia (Caron et al., 1979); it is able to introduce superhelical turns into circular relaxed SV40 D N A but there is no evidence that it binds to m i t D N A either in vitro or in vivo.

In our a t tempt to characterize further the Xenopus laevis mi tDNA-pro t e in complex we have found a protein which has a high affinity for D N A . In this report the purification of this protein is described, some of its properties charac- terized and strong evidence that it is associated with m i t D N A in vivo are presented.

Material and Methods

Oocytes. Whole ovaries were taken from adult female Xenopus laevis and cut into pieces in Barth's medium as modified by Gurdon (1968). The animals were purchased from STACEL France.

Mitochondrial Extract. All operations were carried out in the cold. The oocytes were homogenized in 0.25 M sucrose, 10 mM Tris-HC1 pH 7.5, 1 mM EDTA, 0,1% bovine serum albumin (BSA).

0009-5915/81/0082/0583/$02.20

584 M. Barat and B. Mignotte

After two low speed centrifugations (1,000 g for 10 min) mitochondria were pelleted at 10,000 g for 20 rain then purified on a 0.8 M-2 M linear sucrose gradient containing 10 m M Tris-HC1 pH 7.5, 1 m M EDTA, 0.1% BSA. After centrifugation at 70,000g for 2 h the mitochondrial band was collected, diluted with two volumes of 10 m M Tris-HC1 pH 7.5, 1 m M EDTA and pelleted at 10,000 g for 20 mn. The pellet was resuspended in the diluting buffer containing 1 m M phenylmethylsulphonyI fluoride (PMSF) and sonicated at 0 ~ C for four periods of 30 s with i rain cooling period between each. D N A was removed either by deoxyribonuclease treatment in the presence of 10 m M MgCI> 2 m M CaClz or by centrifugation. This latter procedure was generaly preferred because it avoided the need for extensive dialysis necessary to remove the divalent ions needed to inactivate the enzyme. In this case the sonicated extract was made to a final concentration 2 M NaC1, stirred for 30 mn in the cold, centrifuged at 12,500 g for 15 min to eliminate particulate debris and then at 60,000 g for 16 h. The clear supernatant was used as the crude mitochondrial extract.

MitDNA-Complexes. The isolation of these complexes was done according to Pinon et al. (1978) with two minor modifications. Spermidin was omitted and 2% Triton XI00 was used as detergent.

DNA Cellulose Chromatography. D N A cellulose was prepared with native or heat denatured calf thymus D N A according to the combined methods of Li tman (1968) and Alberts et al. (1971). The columns were run in the cold. The protein solution to be chromatographed (free of DNA) was dialysed against 20 m M Tris-HC1 pH20 o 7.5, 50 m M NaC1, 1 m M EDTA, 2 m M 2-mercaptoetha- nol (buffer A), 10% glycerol and adsorbed on the column which had been equilibrated with the same buffer just before use. After washing the column with 5 bed volumes, the column was eluted stepwise by increasing the ionic strength from 0.1 M to 2 M NaC1. The different eluted fractions, after appropriate dialysis, were either lyophilized or kept at - 2 0 ~ C in buffer A with 60% glycerol.

DNA Binding Assay. The nitrocellulose filters (0.45 gm pore sized BA85, Schleicher and Schfill) were boiled 20 rain in distilled water, then soaked in buffer A (pH25 o 7.9), 5% glycerol and 1% DMSO. Before use they were rinsed in the same buffer except that D M S O was omitted. Routinely the assays were performed as follows: native or heat denatured (0.03 gg) E. coli 3H-DNA (570,000 cpm/ gg) was incubated with the protein solution in buffer A, 5% glycerol and 50 gg/ml BSA (total volume 0.2 ml) at 25 ~ C for 30 rain. The reaction mixture was then filtered through a nitrocellulose filter prewashed with 10 ml of the binding buffer at a flow rate of about 3 ml/min. The filter was then washed with 10 ml of the same buffer and dried; the radioactivity was determined in toluene PPO-POPOP scintillation solution. Control filters without protein solution were routinely carried out in each case.

Velocity Sedimentation Analysis. The protein and sucrose solutions were made in 20 m M Tris-HC1 pH 7.9, 1 m M EDTA, 2 m M 2-mercaptoethanol and the indicated NaC1 concentration. 250 gl of the protein solution were overlayered on a 5 to 20% (w/w) sucrose gradient and centrifuged at 200,000 g for 18 h at 4 ~ C. As necessary the collected fractions were diluted in order to not exceed 200 m M NaC1 in the D N A binding assay. The markers were run in a parelM gradient; E. coli alkaline phosphatase activity (EC 3.1.3.1) was measured by the colorimetric assay of Engs t r6m (1964), the distribution of BSA and horse cytochrome c were determined spectrophotometricalty at 280 nm and 410 nm respectively.

Protein Eleetrophoresis. Protein electrophoresis was performed on 10% polyacrylamide slab gels containing 0,1% Na Dod SO4 using the discontinuous buffer system of Laemmli (1970).

Electron Microscopy. The technique was adapted from Inman (1970). Samples to be analysed were made to final concentration 80 m M Tris-HCl, 8 m M EDTA pH 7.2, 40% formamide. The mixture was left in the cold for 10 min and then cytochrome c 0.01% was added. After 10 rain in the cold the samples were spread on twice distilled water and picked up on a carbon coated mica disk; the disk was rinsed in ethyl alcohol and dried under a lamp. After rotary shadowing with plat inum-pal ladium the samples were floated-off the mica and picked up on electron microscope grids.

Fig. 1. NaDodSO4 polyacrylamide gel electrophoresis pattern of a mitochondrial extract. Lanes b, d, e, f: fractions eluted from the DNA cellulose column at 0.1, 0.2, 0.6, 1 M NaC1; lane a: markers (BSA, RNase, cytochrome c); lane c,g: X. laevis erythrocyte histones

Fig. 2. Electrophoretic comparison of: mitochondrial acidic extract (lane a); purified 12,500 d protein (lane b); X. laevis histones (lane c); markers: DNase (lane d), and BSA, RNase, cytochrome c (lane e)

586 M. Barat and B. Mignotte

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Fig. 3. Retention of E. coli 3 H-DNA on membrane filters. D N A binding activity was assayed in 0.2 M NaC1 with heat denatured D N A at 0 ~ C o - - o and at 25 ~ C. o - - e ; with native D N A at 0 ~ C A zx and at 25 ~ C A - - A

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Fig. 4. Effect of adding increasing amounts of protein to DNA. D N A binding activity was assayed in 0.2 M NaC1. o - - o Heat denatured D N A , zx - - zx native D N A

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Fig. 5. Effect of pH on the D N A binding activity. D N A binding activity was measured in PO4 buffer under pH 7 and in Tris-HC1 buffer at higher pH. 0 0 Heat denatured D N A , z~ - - z~ native D N A ; open symbols PO 4 buffer, full symbols Tris- HC1 buffer

Results

Isolation of the DNA Binding Protein. The mitochondr ia l extract was adsorbed on a double-stranded D N A cel lulose co lumn at 50 m M NaC1. The different fractions eluted at 0.1, 0.2, 0.6, 1 and 2 M NaC1 were dialysed, lyophi l ized and run on a N a D o d SO+ polyacrylamide gel; their protein patterns are shown on Figures 1 and 2. In the fraction eluted at 1 M NaC1 one major band was found at 12,500 daltons with, somet imes , a jo int band at 30,000 daltons. In

A Mitochondrial DNA Binding Protein 587

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Fig. 6. Effect of divalent cations on the DNA binding activity measured in the presence of various concentrations of CaC12 (zx--zx) or MgC12 (A--A). DNA was native

Fig. 7. Effect of NaCI concentration on DNA binding activity. Background was substracted for each concentration, o - - o Heat denatured DNA, z~--zx native DNA

Fig. 8. Velocity sedimentation analysis. Protein solution was sedimented through a 5 20% (w/w) sucrose gradient in 1 M NaC1. DNA binding activity was assayed with a mixture of native and heat denatured 3H-DNA. Markers refer to E. coli alkaline phosphatase (6,3), BSA (4,4), horse cytochrome c (2,1)

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the fract ion eluted at 2 M NaC1 there was no visible band. If the proteins no t adsorbed on the double-s t randed D N A cellulose co lumn were passed over a single-stranded D N A cellulose co lumn no detectable protein was eluted by 1 or 2 M NaC1. The 12,500 d protein was no t retained on a D E A E cellulose co lumn eluted with 20 m M K phosphate pH 7.5 indicat ing that it is rather basic. However, it bands at a posi t ion clearly different from the Xenopus laevis erythrocyte histones run on the same gel (Fig. 2). This protein is no t highly represented among the total mi tochondr ia l soluble protein nor is it present in the acidic mi tochondr ia l protein extract (0.25 M H2SO4). A rough es t imat ion indicates that it represents no more than 0.02% of the proteins loaded on the column. In one part icular experiment mi tochondr ia were treated with digi- ton in (0.2 mg/mg protein) which had been shown very efficient in removing the outer m e m b r a n e of Xenopus mitochondr ia (Brun, submit ted) ; the 12,500

588 M. Barat and B. Mignotte

Fig. 9a and b. Reconsti tution experiment. Purified mi tDNA and protein were mixed in a 1:1 ratio. See text for details. Electron micrographs of m i tDNA molecules with the single-stranded fiber of the D loop covered with protein. Bars represent 0.1 gm

A Mitochondrial DNA Binding Protein 589

Fig. 10. Electrophoretic comparison of the mitochondrial extract and purified mitDNA protein complexes. Fraction eluted at 1 M NaC1 from DNA cellulose. Lane c: mitDNA-protein complex; lane d: mitochondrial extract; lanes a, b, e refer to mitochondrial acidic extract, markers (BSA, RNase, cytochrome c) and X. laevis histones

d polypeptide was still recovered f rom the particular fraction in the usual amount .

In vitro DNA Binding Properties. All these experiments have been done with a 12,500 d protein solution obtained by ch roma tog raphy on D N A cellulose o f the proteins not adsorbed on D E A E and kept at - 2 0 ~ C in 60% glycerol. In this case the protein is pure as judged by Na D o d SO4 gel electrophoresis. The protein binds to single and double-s t randed E. coli D N A with apparent ly the same efficiency. The binding reaction is very rapid, the max imum retention obtained was a little slower at 0 ~ than at 2 5 ~ but at bo th temperatures a plateau is reached in less than 10 rain (Fig. 3). The effect o f adding increasing amounts o f the protein to D N A is shown on Figure 4, there is no cooperat ive effect for either D N A . N o op t imum p H was found for binding activity (Fig. 5). The addit ion to the assay mixture o f Ca + + or Mg ++ in the range 0.5-2 m M has no real effect on the binding efficiency (Fig. 6). M a x i m u m retention to the filter was obtained at 200 m M NaC1 (Fig. 7).

Velocity Sedimentation Analysis. F r o m sucrose gradients performed in 0.5 or 1 M NaC1 a sedimentat ion coefficient o f 4S-equivalent to a molecular weight

590 M. Barat and B. Mignotte

Figs. 11 and 12. Electron micrographs of mitDNA-protein complexes. Fractions of the purified complexes were spread without fixation. For details see text. Bars represent 0.1 gm

of abou t 50,000 dal tons was found indicat ing that in its native form this prote in may be a te tramer (Fig. 8).

Interaction with Xenopus laevis mi tDNA. The prote in was mixed with purified m i t D N A in low salt condi t ions and incubated at r oom temperature for 30 rain. The m i t D N A used conta ined an average of 50% D loop structures. The ratio

A Mitochondrial DNA Binding Protein 591

of protein to D N A was approximately 1 : 1. After fixation with buffered glutaral- dehyde to a final concentration of 0.15% the preparation was visualized by the Inman spreading technique. In these conditions the protein seems to bind preferentially to the single-stranded part of the D loop structure (Fig. 9).

MitDNA-Protein Complex Analysis. In order to determine if this protein was associated to m i tDNA in vivo the proteins f rom purified m i t D N A protein complexes were run onto a D N A cellulose column as previously described. The electrophoresis pattern of the fraction eluted at 1 M NaC1 presented on Figure 10, clearly shows a band corresponding to the 12,500 d polypeptide.

592 M. Barat and B. Mignotte

Other proteins eluted at lower ionic strength were also found associated with the purified complexes (in preparation). The comparatively low amount of this protein recovered from mitDNA protein complexes and the apparent preferential binding to the single-stranded part of the D loop in reconstitution experiments led us to look for replication complexes. MitDNA protein complexes were extracted from oocytes of young females which contain a high percentage of D loop structures (Callen, personal communication). Fractions of the purified complexes were directly analysed by the previously described EM technique without any fixation in order to optimize the spreading of the D loop structure. In these conditions the beaded structure is not preserved; mitDNA molecules are "naked" except the single-stranded fiber from the D loop which is covered with protein (Figs. 11 and 12.)

Discussion

In this report we describe a DNA binding protein, purified to apparent homoge- neity, from Xenopus laevis oocytes mitochondria. Its localization inside the organelle and its association with mitDNA molecules in vivo are supported by several lines of evidence. (i) This protein is isolated from highly purified mitochondria even if they have been treated with digitonin. (ii) Its electrophoretic mobility on Na Dod SO4 polyacrylamide gel is different from that of Xenopus laevis histones (MW= 12,500 daltons) and it is polymeric in its native form, perhaps a tetramer (4S sedimentation coefficient). (iii) It is recovered from purified mitDNA-protein complexes among the proteins associated with DNA.

This protein seems to be tightly associated with mitDNA by ionic bonds as the binding can withstand the drastic conditions used for the E.M. psreading technique including 40% formamide. Its high affinity for DNA is also shown by the ionic strength of 1 M necessary to elute it from DNA cellulose.

The electron microscopic analysis by the Inman technique of both purified mitDNA-protein complexes und products of reconstitution experiments between mitDNA and the described protein yielded very similar pictures. In both cases only the single-stranded part of the D loop structure is covered with protein; the length of the fiber is reduced 1.5 times on the average (maximum 2.5). By complexing to single-stranded DNA, DNA binding proteins have been shown either to decrease its length like E. coli DBP I (Sigal 1972) or to increase it like the 32 protein (Delius, 1972). Even though no difference between single and double-stranded DNA has been found in DNA binding assay using E. coli DNA, the apparent preferential binding is very likely not sequence specific because the size of the D loop corresponds to 9% of the DNA molecule (about 1,500 nucleotides). It is not yet ruled out that this protein is unable to bind to double-stranded superhelical mitDNA; for example, in the reconstitution experiment, compact structures could have been counter selected by the used EM technique. Also a ratio of protein to DNA higher than 1 : 1 may be needed. However, we do not think that this protein is responsable for the packing of mitDNA ; other proteins have been found associated with mitDNA (in prepa- ration), they are bound more weakly to DNA, which is in agreement with the instability of the mitDNA-protein complexes.

A Mitochondrial DNA Binding Protein 593

O n the basis o f o u r resul ts it w o u l d be h a z a r d o u s to p r o p o s e a prec ise

f u n c t i o n fo r this p ro te in . W e sugges t t ha t it s tabi l izes the s ing l e - s t r anded D N A

f iber o f the D l o o p s t ruc tu re a n d p ro tec t s it f r o m nuc lease a t t ack . A l t h o u g h the exac t f u n c t i o n o f D l o o p s t ruc tu res has n o t yet b e e n es tab l i shed , the i r

a c c u m u l a t i o n at p a r t i c u l a r s tages o f oogenes i s , (Cal len , p e r s o n a l c o m m u n i c a t i o n )

in the absence o f m o r e a d v a n c e d r e p l i c a t i o n f igures c o u l d ind ica te a r e g u l a t o r y

func t ion . A poss ib le s t i m u l a t i o n o r i nh ib i t i on o f r ep l i ca t i on a n d / o r t r a n s c r i p t i o n

has to be s tudied.

Acknowledgements. Financial support was provided by the Centre National de la Recherche Scienti- fique, ATP no. 4025 and L.A. 86. Electron microscopic work was done in G.Y. Brun's laboratory. We thank Dr. G.M. Brun for a generous gift of E. coli 3H-DNA.

References

Alberts, B., Herrick, G. : DNA cellulose chromatography. Methods in enzymology, (S.P. Cotowick, and N.O. Kaplan, eds.), vol. 21D, pp 198-217. New York: Academic Press 1971

Caron, F., Jacq, C., Rouvi~re-Yaniy, J. : Characterization of a histone-like protein extracted from yeast mitochondria. Proc. nat. Acad. Sci. (Wash.) 76, 4265-4269 (1979)

Delius, H., Mantell, N.J. : Characterization by electron microscopy of the complex formed between T4 bacteriophage gene 32-protein and DNA. J. molec. Biol. 67, 341-350 (1972)

Engstr6m, L. : Studies on Bovine-liver alkaline phosphatase, purification, phosphate incorporation. Biochim. biophys. Acta (Aust.) 92, 71-78 (1964)

Gurdon, J.B.: Nucleic acid synthesis in embryos and its bearing on cell differentiation. Essays Biochem. 4, 25-68 (1968)

Inman, R.B., Schn6s, M. : Partial denaturation of thymine and 5-Bromouracil containing 2DNA in alkali. J. motec. Biol. 49, 93-98 (1970)

Kuroiwa, T., Kawane, S., Hizuma, M. : A method of isolation of mitochondrial nucleoid of Physa- rum polycephalum and evidence for the presence of a basic protein. Exp. Cell Res. 97, 435-440 (1976)

Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.) 227, 680-685 (1970)

Litman, R.M. : A deoxyribonucleic polymerase from Micrococcus luteus (Micrococcus lysodeikticus) isolated on deoxyribonucleic acid cellulose. J. biol. Chem. 243, 6222-6233 (1968)

Olszewka, E., Tait, A.: Mitochondrial chromatin in Paramecium aurelia. Molec. gen. Genet. 178 453~458 (1980)

Pinon, H., Barat, M., Tourte, M., Dufresne, C., Mounolou, J.C.: Evidence for a mitochondrial chromosome in Xenopus laevis oocytes. Chromosoma (Bed.) 383 389 (1978)

Sigal, N., Delius, H., Kornberg, T., Gefter, M.L., Alberts, B. : A DNA unwinding protein isolated from Escherichia coli: its interaction with DNA and DNA polymerases. Proc. nat. Acad. Sci. (Wash.) 69, 3537-354I (1972)

Van Tuyle, G.C., McPherson, M.L.: A compact form of rat liver mitochondrial DNA stabilized by bound proteins. J. biol. Chem. 254, 6044-6053 (1979)

Received October 7-November 6, 1980 / Accepted by W. Beermann Ready for press November 28, 1980

Note Added in Proof:

Filter binding assays made with native and heat denatured linearised pBR 322 plasmid DNA, and competition experiments indicate that the apparent affinity for the native DNA was probably due to short single strand stretches.