Purification and Properties of Deoxyribonucleic Acid Polymerase … · 2003-02-01 · 4906 DNA Polymerase from Rat Liver Mitochondria Vol. 243, No. 1s DNA was quantitatively determined

THE JOURNAL OF BIO~GICA~~ CHEMISTRY

Vol. 243, I’io. 18, Issue of September 25, pp. 4904-4916, 1968 Printed in U.S.A.

Purification and Properties of Deoxyribonucleic Acid

Polymerase from Rat Liver Mitochondriae

(Received for publication, April 26, 1968)

GEORGE F. KALF$ AND JOHN J. CH’IH

From the Department of Biochemistry, Je$erson Medical College, Philadelphia, Pennsylvania i9107

SUMMARY

These studies report the isolation of a DNA polymerase from rat liver mitochondria. The enzyme has been purified 22-fold by differential ultracentrifugation, ammonium sulfate precipitation, and column chromatography on DEAE- cellulose. The partially purified enzyme manifests all of the requirements shown by the known DNA polymerases of mammalian nuclei and bacteria.

The enzyme has an optimal pH of 7.5, and requires Mg+f (12 mM) and the presence of all four deoxyribonucleoside triphosphates. The reaction is inhibited by pyrophosphate and by the addition of deoxyribonuclease but not by ribonu- clease.

Enzyme purified in this manner is free of nuclear DNA polymerase, terminal addition enzyme, and DNase. The enzyme shows an absolute requirement for added DNA template which can be furnished rather specifically by native, double-stranded, circular, mitochondrial DNA.

Furthermore, the labeled DNA was shown to be mitochondrial in nature by virtue of the fact that it had the same buoyant density in CsCl as unlabeled, highly purified, mitochondrial DNA and the fact that it was renaturable after heat denaturation, a property shown by mitochondrial DNA but not by mammalian nuclear DNA.

The product of the reaction was shown to be a double stranded replica of the mitochondrial DNA template on the basis of autocatalytic synthesis by the enzyme with the use of nonsaturating levels of mitochondrial DNA template. A 3.5-fold synthesis of new DNA was achieved.

The general occurrence of a unique DNA in mitochondria has been well documented (1, 2) and a biological role for this DNA

* These investigations were supported by United States Public Health Service Predoctoral Fellowship IFl-GM-32,992 to J. J. Ch’ih and by Research Grants 377A and 377B from the American Cancer Society. The results were taken, in part, from the Ph.D. dissertation presented by J. J. Ch’ih to the Graduate School, Jefferson Medical College, 1968.

$ This work was carried out during the tenure of an Established Investigatorship from the American Heart Association, Inc.

has been established by the demonstration that these organelles can effect the DNA-dependent synthesis of RNA (3-6) and protein (7, 8) in vitro.

Mitochondria also are capable of the incorporation of DNA precursors in vivo (I), and this incorporation appears to be inde- pendent of the mitotic cycle of the cell (1). Furthermore, the turnover of mitochondrial DNA is usually higher than that of nuclear DNA (9).

While our studies were in progress, workers in several laboratories reported on the ability of intact mitochondria from rat liver (10, II), yeast (la), and the mold, Phyrasum polycephalum (13), to carry out the incorporation of deoxyribonucleoside triphosphates into mitochondrial DNA in titro. The character- istics of the incorporation reaction shown by isolated, intact mitochondria are similar to those shown by the DNA polymerases of mammalian nuclei (14) and bacteria, and suggest that mitochondria possess a DNA polymerase.

In this paper, we wish to report the purification and characterization of such a DNA polymerase from rat liver mitochondria-free of contamination by the nuclear polymerase.

EXPERIMENTAL PROCEDURE

dfaterials

Animals and Tissues

Female Wistar rats (150 to 200 g) were used throughout this study; they were fasted overnight before decapitation. Escher- i&a coli cells, strain W, harvested in midlog phase, were purchased from General Biochemicals.

Chemicals and Reagents

Enzymes-Pancreatic DNase I, pancreatic RNase, lysozyme (chicken egg white), bovine spleen phosphodiesterase, and micro- coccal nuclease were purchased from Worthington. Pyruvate kinase was obtained from Sigma.

Isotopes-dATP-8-14C (15.3 or 28.4 mC per mmole), ATP-& 1% (10 mC per mmole), dTMP-2-l% (13.5 mC per mmole) and dTTP-2-W (49 mC per mmole) were purchased from Schware BioResearch.

Biochemicals-Sigma was the source of the unlabeled ribonucleoside triphosphates, the deoxyribonucleoside mono- and triphosphates, Tris, phosphoenolpyruvate, RNA (yeast), and

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DSd (calf thymus) DSli preparations obtained from com- mercial sources wcrc furthrr purified by alcohol 1)rccipitatioll before use. (‘csium chloritlc (optical grade) n-as 1)urchawd from Gullarrl-S~hlcsinger (‘hemica1 Corl)oration, Long Island, New York. Sc1)hudcs G-l 00 dextran gel was obtainrd from Pharmacia Fine Chemicals, Inc., :nld I)EAE-sclcctncel ion OS- change cellulost \V:IS :I product of the Carl Schleichcr and Schurll Conrl):my. The I)E.\E-cellulose was prepared for UPC by the procedure of Pctcrson and Sobrr (15).

2,5-T>iphrn~losazole (PPO), 1,4-bis[2-(5-pheny1os:lxolyl)]ben- zenc (1’OPOP), and Hyaminc 10X VW~ from Packard Instrument Company; Surlcar-Chicago Solubilizcr from Xuclcxr-Chicago; and cellulose awlat c filter discs (0.45 p) from the Millipore Filtrr Corlwration.

Actilwmycin 1) WV the gift of T>r. I)nvid Hendlin of Merck Sharp and Dohmc R~scarch Laboratories, and phlcomycin was the gift of 1)r. Alcsandcr Gourevitch of Bristol Laboratories, Inc., Syracuse, Sew York.

Neihods

Isolation OJ” Intact Subrellular Components and Related

Prt7KuYltions

Preparaiion of Rat Liver Illitocl~ondria-Thc method of O’Brien and Kalf (16) was used. All operations were Iwrformed at O-4”. 1Vith t,his 1)roccdure the yield of mitochondria was 6 to 9 mg of nlitocholldrial 1)roteill per g of liver.

Preparation of Calf Lioer dlitochondria-~Iitochontll~lria were prepared from calf lirtr by the 1)rocedure described for the prepa- rat,iou of rat liver mitochondria rrcept that the calf liver \vas millcctl by pnssagc through an ice-cold cltctric mcat grindrr and the homogenate wxs prrparcd 11). blrnding the miucc for 9 SW in 10 volumes of isolation medium in a Waring Mendor. The isolation medium was 0.34 M sucrose-2 nine Tris, pH 7.4.

Preparation of Intact Rat Liver n’wlei-A modification of the method of Chauvcau, ~IoulB, and Rouillcr (17) was used. Livers mere quickly ewiscd and immersed in isolation medium (0.34 RI

sucrose-2 mx Tris, 1)H 7.4) at 0”. The tissue TT--as then weighed, minrctl n-ith scissors, and homogcnizcd ill 10 volumes of isolation medillm by six 1)c‘stle strokes in a standard, hand~opcratcd Ten l<rocck tissur grinder.

The homogenate !v:ls cclltrifugcd at 1,020 X g (2,500 rl)n’) for 10 mill ill a Sorvall GSA rotor to setlimcnt Iluclri. The rrudc pcllct was rchonlogcllizcd in 10 volumes of 2.2 hI surrosc, and the homogcuate was filtcrcd through six lnycra of washed cheesecloth alltl sul)jectcd to cc,Iltrifugation in a Spinro So. 30 rotor at 38,400 x g (21,000 r1)m) for 1 hour to harvest thr nuclear pellet. The 1)cllct I\-as rcsuslwndcd ilr 2.2 M sucrose and cclltrifugcd as abOW. The final nuclrar prrlwation contained virtually 110 rrtl blood ~~~~11s and rssclltially no mit ocholldria, as wrificd by 1)h:we microsco1)y.

Preporation of .Vuclei and dlitoclrondria from the Same Ilo- nrogenrrtc-IVhrn it was ~~rccssnr~ to ibolate both nuclri :uld mitochondria from tllc same homogenate, the methotl 1)reviouslJ descrihctl for the isolation of nlitorholldria n‘as followed rscept that the pcllct, obtained at”& rcntrifugation of the homogenate at I ,020 x g was not discardrd but n-as treated according to the prowdure drscribrd for ihc 1)rcl)aration of rat liver lrwlci.

Isolation of Various DA’:1 s-;\Iitochondrial DNh was prr- pad by the mcthotl of Kalf al\d Gr&e (18). Xuclcnr DSA wts prcparctl from purified nuclei by the method used to estrwt

DN=1 from mitochondria so that the DNA would be cxposcd to identical conditions. DNA was prrparrd l’rom l?. roli cells 1,) thr nlcthod of Snlith and I3urtoll (19).

Hmt Denaturation of DA-A-Thermally dniaturrd DX.As were prrparcd by following the procedure lmblishcd by Kay, Simmons, and Dounce (20).

Estimation of Purity of Intact SubceIlular Components and

Related Assays

Phase JficroscoplJ-l’rc1)nr:rtiolls of fivr-times washed mitochondria or nuclei were esnmincd routillcly by phase nlicroacol)> to ascertain the level of contanlillation by red blood cells, whole cells, and cell debris.

Ztochondria were stained with Janus grcrn 1% to facilitate visualization and to establish the lack of nuclear collt:\l~lin:ltion. One drol’ of mitochondrial swpcnsion (10 mg of mitochondrinl protein 1)cr ml) was stained with 0.01 “/;, Janlls green 1% and csam- incd by 1)h:tse mirroscol)y.

Electron .Ilicroscopy-The nlitochondrial lwllrt was fixed in newly distilled glutaraldehydc, 67; glutaraldehyde in 0.28 nr sucrose-O.01 ar potassium phosphate, pH 7.4. Fixation ill the above glutaraldehpdc solution was extended for 1 hour at 4”.

Postfixation was carried out, for 1 hour at room temlwrature in Mllonig phosphate-buffered osmium solution. The pcllcta were then dehydrated in increasing concentrations of ethanol at 4” and embedded in Maraglas (Marblette Corporation, Long Island City, New York) according to the procedure of Erlandson (21).

The sections of rmbedded material were esamined on naked copper grids with an RCh EMU-3D clcctron microscope at 100 kv.

We are indebted to Doctor Charlrs Rosa of the Department of Anatomy, Jefferson Medical College for carrying out the electron microscopy.

Bacterial Confawination-Throughout the investigation the following precautions n-ere carried out t,o minimize the possibility that incorporation of radioact,ivcly labeled compounds by intact mitochondria and enzyme preparations was due to the presence of contaminating bacteria. The abdomen of the exlwrimental animal was sterilized with 70% alcohol brfore rcmovul of the liver; all solutions were prepared immediatrlg before use; actual bacterial contamination n-as checked from time to time b? plating a sample of the mitochondrial 1)rrparation or cliz~-nit lw1xwation and the incubation mixture (before and aft.rr incubx- tion) on blood agar and incubating the plate for 24 to 48 hours at 3i”. Over a large number of 1)reparations, colon\- counts varied bctwecn 540 and 870 colonies prr mg of niitochondrial protrin. Furthermore, the number of bacteria did not increase durillg the course of the 30.min incubation.

The role of bactcrinl contaminnt ion has brcn discussrd in the literatuw and csrludcd as an important contributioli to mitochondrial illcorl)oration mrnsurcd by differrnt rs1wimcntal desiglrs. It was co~~rlutlcd that thrrc was 110 correlation Fvhatso- ever bctn-rcn thr amount of contaminating bacteria and the mitochondrial incorporafing activity (22-26).

d nalytical Assags

Chemical Deter,,,inatio,ls-I’lotciii was dctrrminrd rithrr I,! the biurrt method (27) or by thr method of Lowry et nl. (28). In sonic instanrrs, 1)rotein collrrtltralion was drtrrlnillrd h1w~ tro1~hotomrtric:~lly iu the Zeiss 1’AlQ II s1,cc~troI)hotorlicter b>- mrasurillg the absorl)tioll of light at 280 111~ alltl 260 nip.


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4906 DNA Polymerase from Rat Liver Mitochondria Vol. 243, No. 1s

DNA was quantitatively determined by Burton’s modification (29) of the diphenylamine method with calf thymus DNA as reference, or spectrophotometrically by measuring A260 and then using 0.020 cm2 per pg as the specific absorbance of DNA.

Enzyme Assays-DNase I was assayed by the method of Kunitz (30). DN.4 polymerase was routinely assayed by determining the amount of radioactively labeled deoxyribonucleoside triphosphate incorporated into acid-insoluble, DNase-sensitive product or, more specifically, in some experiments into mitochondrial DNA.

Reaction mixtures were prepared in chilled, 12-ml, heavy duty, conical centrifuge t,ubes. Generally the standard incubation mixture contained DNA\ template (type and amount of template are specified for each experiment), three unlabeled deoxyribonucleoside 5’.triphosphates (30 mHmoles each) and the fourth deoxyribonucleoside 5’-triphosphate furnished as the radioactively labeled one (24 mFmoles of dTTP-2-r4C, specific act,ivity of 49 mC per mmole, or 50 mpmoles of dATP-8-14C, specific activity of 28.4 mC per mmole), a nucleoside triphosphate-gen- erating system composed of phosphoenolpyruvate (3.75 pmoles) and pyruvate kinase (10 pg) to prevent the hydrolysis of dATP- 8.14C due to the presence of ATPase within the mitochondrion, MgCIZ (5 ~molcs), 2-mercaptoethanol (770 mpmoles); Tris-HCl buffer (50 pmoles, pH 7.5), and 40 to 100 pg of enzyme protein preparation such as that obtained from the crude extract, 50 to 70% ammonium sulfate fraction, or Peak B, as specified in each experiment. The final volume was 1 ml, and the tubes were incubated for 30 min at 37” unless otherwise noted.

In the early experiments, when only the incorporation of labeled DNA precursors by intact mitochondria was studied, a sample of a mitochondrial suspension (1 mg of mitochondrial preparation) was used in place of the enzyme preparation. These incubatioiis were carried out in 25.ml Erlenmcyer flasks instead of conical centrifuge tubes.

At the end of the appropriate incubation period, the reaction was stopped by the addition of an equal volume of ice-cold 10% trichloracetic acid, and 500 pg of bovine serum albumin, 50 pg of native calf thymus DN-4, and 50 m&moles of unlabeled dATP were added as carriers. The mixture was allowed to precipitate in the cold for 20 min, after which the insoluble precipitates were subjected to the procedure for the determination of radioactivity.

The activity of DNA-dependent RNA polymerase present in the DN.4 polymerase preparations was measured by the procedure described for DNA polymerase except that i4C-labeled ATP (50 mpmoles; specific activity, 10.1 mC per mmole) and a mixture of GTP, CTP, and UTP (60 mpmoles each) were substituted for the deoxyribonucleoside triphosphates. The reaction was stopped in the same manner as for the DNA polymerase assay, except that. 50 pg of yeast RNA and 50 mpmoles of ATP were used as carriers.

Determination of Radioactivity-Material containing acid- insoluble radioactivity was washed three times with ice-cold 5y0 trichloracetic acid, three times with ether-ethanol (3:1), and finally with ether. The precipitates were then dissolved in 0.1 N NaOH. (This step was omitted in the assay of RNA polymerase.) An aliquot of this alkaline solution was removed for protein determination to correct for the loss of materials during washing. The material was reprecipitated from the NaOH by the addition of ice-cold 50% trichloracetic acid to a final trichloracetic acid concentration of 5T0. Removal of trichloracetic acid from the pellet was accomplished by washing with ether-ethanol.

The final precipitates were dissolved in 0.5 t,o 1.0 ml of Hyamine 10X or Nuclear-Chicago Solubilizer, and the solution was warmed to 50-60” to faciliate dissolution. To this solut.ion were added 10 ml of scintillation medium of the following composition: 2,5- diphenyloxazole, 4.0 g; 1,4-bis[2-(5-phenyloxazolyl)]benzene, 100 mg; and toluene, 1000 ml.

In certain experiments, material containing acid-insoluble radioactivity was recovered from the incubation mixture by col- lection on lllillipore cellulose acet,ate discs. To facilitate the precipitation of small amounts of material in trichloracetic acid, 100 pg of bovine serum albumin, 50 pg of calf thymus DNA, and 50 mpmoles of unlabeled dATP were added as carriers. The Millipore discs containing the radioactive samples were washed 10 times with 2.5.ml portions of ice-cold 5y0 trichloracetic acid and then placed in counting vials. The precipitate adsorbed on the disc was dissolved in 0.5 ml of Nuclear-Chicago Solubilizer for 3 hours at room temperature. Scintillation medium (10 ml) was then added to this mixture and determination of the radioactivity was carried out.

Radioactivity was determined with a Nuclear-Chicago Ytark I scintillation system. The background was 20 to 25 cpm, and the counting efficiency for r4C was 76 to 857;. Quenching was corrected for by using the channel ratio met,hod with an external standard. In the experiments wit,h intact mitochondria the sample radioactivity was generally only twice background, a problem confronting workers studying incorporation into intact mitochondria. Therefore, it was necessary in these esperiments (Table II, below) to count the sample for 15 hours. However, experiments carried out with crude extract or enzyme fractions showed sample counting rates 10 times background, and all determinations of radioactivity throughout this part of the investigation were carried out to an accuracy equivalent to 1 y0 standard deviation by collecting 10,000 counts from all samples.

Cesium Chloride Density Gradient Cenfrifugation-Sedimenta- tion equilibrium studies with the use of a cesium chloride buoyant density gradient were carried out according to the method of Vinograd (31). A stock CsCl solution (~~~0, 1.9077) was prepared by dissolving 13 g of optical grade CsCl in 7.0 ml of 20 m&f Tris-HCl buffer, pH 8.5. To 1.96 ml of this solution was added enough of a solution of 0.54 ml of DNA in 0.15 RI NaCl-0.015 M

sodium citrate to obtain the desired density at, 25” of 1.710 g cm-3.

Ultracentrifugation was carried out in polyallomer tubes in the Spinco SW 39 swinging bucket rotor. The centrifuge tube was tilled with 2 ml of the DNA-CsCl solution and overlayered with 2.5 ml of light mineral oil to prevent evaporation of the solution and collapse of the tube. Centrifugation was carried out at 30,000 rpm for 88; hours at 25”. At the end of the run, decelera- tion was allowed to proceed without braking. The tubes were removed from the rotor and individually mounted in a rubber stopper sleeve on a ring stand. The DNA was recovered from the gradient by piercing the bottom of the tube with a sharp needle and collecting 4-drop fractions. The refractive index (ni5) of each fraction was determined with a Bausch and Lomb Abbe 3L refractometer, and t.he density of each fraction was calculated (31).

Purification of Jf itochondrial DNA Polymerase

Crude Extract-Jlercaptoethanol was added to all buffer sys- tems used in the purification procedure since higher enzymic


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activities were obtained whenever it was used. Unless otherwise noted, all operations were carried out at O-4”.

In a typical purification, rat liver mitochondria (5 times washed; 1,025 mg of protein) were suspended in 20 mM potassium phosphate buffer, pH 7.2, containing 1 mM 2-mercaptoethanol and 1 mM EDT.1 (Buffer A) to a final protein concentration of 15 to 30 mg per ml. The suspension was subjected to three cycles of alternate freezing in acetone-solid carbon dioxide and thawing at 20”, followed by extraction at 4” with stirring for 16 hours. The crude extract was prepared by centrifuging the susl)cnsion of disrupted mitochondria at 41,000 x g (25,000 rpm) for 15 min in the Spinco No. 40 rotor and removing the clear supernatant fluid. The crude extract contained approximately 126 mg of protein in 57 ml of extract and was used for further purification of the enzyme.

Preliminary Purijcation by Differential Ultracentrifugation- After centrifugation at 151,925 x g (50,000 rpm) for 6 hours in a Spinco No. 50 rotor, each tube appeared to contain four phases which are partially nucleoprotein in nature. Beginning at the bottom of the tube, these phases are referred to as Fractions I, II, III, and IV. The various fractions were removed with a Past,eur pipette according to the following fixed proportion. Fraction IV, the top 3.0 ml from each tube, generally comprised approximately 307, of the total volume. The volume of Frac- tion III was 4.0 ml; Fraction II was 1.7 ml. The remaining 1.3 ml were drawn off as Fraction I.

As can be seen in Table I, most nucleoprotein appeared to be concentrated in Fraction II. DNA polymerase activity was highest, in Fractions 1 and II. Because of its high protein content,, however, Fraction II was selected for the subsequent ammonium sulfate step.

Removal of Sucleic A-lcid-Fract.ion II (10.5 ml in Buffer A), containing approximately 6 mg of protein per ml, was adjusted to pH 7.7 with KOH and made 2 InM with respect to iMg++ ion. The nucleoprotein complex was degraded by the addition of pancreatic DBase (2.5 pg per ml final concentration) and pancreatic RXase (20 pg per ml final concentration) to Fraction 11 and incubation of the mixture at 37” for 3 hours. At the end of this time, the digest was adjusted to a final volume of 25 ml with Buffer ;1 and chilled to 0” before the addition of ammonium sulfate.

rlnznloniul?z Suljaate Fractionation-Fraction II (25 ml; 60 mg of protein) from the previous step was taken to 30% saturation with ammonium sulfate by the addition of solid ammonium sulfate with constant stirring. The pH was maintained at 7.2 by the dropwise addition of NHIOH. The mixture was stirred for 30 min, and the precipitated protein was collected by centrifugation at 44,149 x g (22,500 rpm) in the Spinco Ko. 30 rotor for 10 min. The precipitated material was discarded and the supernatant fluid was adjusted to 50% saturation. The precipitate was collected and discarded, and again solid ammonium sulfate was added to the clear supernatant fluid to obtain the 50 to 70% ammonium sulfate fraction. After centrifugation the precipitate was dissolved in 5 ml of Buffer A. The solution was freed of ammonium sulfate by dialysis overnight against 100 volumes of Buffer A, with two changes of the dialyzing buffer. The clear amber solution (Fraction AS 50-70) thus obtained contained about 4 mg of protein per ml and had an absorbance ratio, A 280:L4,,,, of 1.48. Experiments with this fraction were per- formed as soon as possible because of a rapid loss of activity over a period of 72 hours. The fraction was usually kept, at O”, and

in 72 hours retained 40% of the activity. Freezing at -20” in the presence of 1 mg of bovine serum albumin per ml protected somewhat; this preparation retained 43% activity over a 6-day period. The addition of glycerol to a final concentration of 30% did not protect the enzyme activity.

DEAE-cellulose Chromatography-A column, 1.2 cm in diame- ter and 18 cm in length, was prepared from DEAE-cellulose, previously equilibrated with 0.02 M potassium phosphate, pH 7.2, and washed with 25 volumes (500 ml) of the same buffer. A sample (4 ml) of Fraction AS 50-70 (3 mg of protein per ml) was applied to the column, which was then eluted successively with 120 ml of 0.02 M potassium phosphate, pH 7.2, 80 ml of 0.06 M potassium phosphat,e, pH 7.2, and finally with 60 ml of 0.06 M potassium phosphate, pH 7.2, plus 0.1 M ammonium sulfate. The flow rate was held constant at 2 ml per min by means of a Buchler polystaltic pump. The eluates passed through a IO-mm quartz cell of a TMC Vanguard 1056 A ultraviolet ana- lyzer, and the absorbance at 278 rnh was recorded. Fractions of 1 ml were collected. The fractions comprising each peak were pooled and the protein was precipitated by addition of ammonium sulfate to 70% saturation. The precipitated protein was dissolved in 20 mM potassium phosphate, pH 7.2, and dialyzed to remove ammonium sulfate.

RESULTS

Incorporation of Radioactively Labeled DN.4 Precursors by Intact Mitochondria in Vitro

Data are presented in Table II which show that intact mitochondria, incubated under conditions generally used in the assay of the known DNA polymerases, are capable of effecting the incorporation of dATI’-‘4C or dTI\IP-‘4C into acid-precipitable material in vitro. This incorporation was found to be linear for at least 1 hour.

The observed incorporation depends upon Mg++ and partially upon the presence of the other three unlabeled deoxyribonucleoside triphosphates (Table II; “dATP-W”). The lability of the system to heating suggests that it is enzymic in nature. Further- more, the radioactivity of the acid-precipitable material was removed completely by exposure to 5% trichloracetic acid at 90” for 15 min, indicating that the labeled nucleotide was incorporated into nucleic acid.

The demonstration of the ability of intact rat liver mitochondria to carry out the incorporation of DNA precursors raises the question of whet,her the observed incorporation is the result of an intramitochondrial DNA polymerase or merely reflects contamination by a small amount of a nuclear system.

So contaminating nuclei were observed by phase microscopy. ,4n inspection of an electron photomicrograph of washed mitochondria, which is presented in Fig. 1, shows that nuclear fragments are virtually absent.

Furthermore, pancreatic DNase, which can digest nuclear DNA completely in an incubation mixture containing mitochondria, had no inhibitory effect on incorporation of either dTTP-2-W or dATP-8-W by intact mitochondria. The imper- meability of intact mitochondria to enzymic attack by DNase has been well documented (3, 4).

The results of the phase and electron microscopy, together with the inability of DNase to inhibit, suggested that the observed incorporation was taking place within the mitochondrion per se.


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4908 DNA Polymerase from Rat Liver Mitochondria

TABLE I

Vol. 243, No. 18

Distribution of protein, nucleic acid, and enzyme activity in fractions obtained by di$erential ultracentrifugation The standard reaction mixture contained the following compo- ethanol; 50 pg of native calf thymus DNA; 3.75 pmoles of phos-

nents, in a final volume of 1.0 ml: 50 mmoles of dATP-S-l% phoenolpyruvate; 1Opg of pyruvate kinase; 50pmoles of Tris-HCI (specific activity, 28.4 mC per mmole) ; 50 mpmoles each of dCTP, buffer, pH 7.5; and 1OO~g of protein of each enzyme fraction. The dGTP, and dTTP; 5 rmoles of MgCln; 0.77 pmole of Z-mercapto- incubation was carried out at 37” for 30 min.

Preparation

Crude extract. _. Differential ultracentrifugation

Fraction IV.. Fraction III, Fraction II Fraction I.

Volume Protein

% total

100

30 40

17 13

100

ho, Ano:Am Nucleic acid Specify activity

% ,.~pnoks dAMP-“C incorfmzted/ntg fir&in

0.74 8-9 31

16 0.54 20 24 0.57 147 50 0.96 3.5-4.0 238 10 1.06 2.5-3.0 214

TABLE II

Factors affecting incorporation of dTMP-‘Y! and dATP-‘4C by intact mitochondria

The incubation mixtures contained Tris-HCl buffer, pH 7.5 (50 rmoles) ; MgCl2 (5 pmoles) ; 2-mercaptoethanol (770 mpmoles) ;

unlabeled deoxyribonucleoside triphosphates, minus the appropriate labeled one (30 wmoles of each) ; dTMP-2-W (40 mmoles;

specific activity, 13.5 mC per mmole) or dATP-8-W (50 mpmoles; specific activity, 15.3 mC per mmole); and 2.0 to 2.5 mg of mitochondrial protein. The final volume was 1 ml. Duplicate

samples were incubated at 37” for 30 min. The incorporation data represent the average of duplicate determinations; only duplicates which agreed within 5% or less were used.

Conditions ‘4C-Deoxyribonucleotide incorporated

dTMP-‘4C Complete system........................ Hot trichloracetic acid washa.. -Mg++. + Phleomycin (5 pg per ml). + Actinomycin D (2 pg per ml)..

dATP-1%

0.85 0.05

0.38 0.40

0.02

Complete system. 1.09 - dCTP., 0.61

- dTTP.. . 0.63 - dCTP, dGTP, and dTTP.. 0.47

+ Actinomycin D (2 pg per ml). . . 0.21 Complete system, heated*.. . . 0.33

0 5yo trichloracetic acid at 90” for 15 min. * The heat treatment was carried out at 95” for 15 min.

The intramitochondrial nature of the incorporation was confirmed in yet another way. Although mitochondria were im- permeable to DNase, it was thought that inhibitors of DNA polymerase such as phleomycin and actinomycin D, which are lipophilic, might penetrate the mitochondrial membrane and suppress the incorporation. The results of these experiments are presented in Table II; they show that phleomycin (5 pg per ml) inhibited the incorporation of dTMP-i4C approximately 53 ‘% and that actinomycin D inhibited the incorporation of both dTMP-14C and dATP-14C.

We also have observed that concentrations of phleomycin (50 pg per ml) which virtually abolish incorporation of dATP or

dThiIP suppress incorporation of ATP only about 15%, indicat-

ing that the antibiotic preferentially inhibits DNA polymerase. The incorporation of deoxyribonucleotides into acid-precipitable material by intact rat liver mitochondria appears to be carried out by an intramitochondrial enzyme system similar to DNA polymerase. Conclusive proof rests with the isolation and characterization of the enzyme.

Partial PuriJication of Enzyme

A summary of a typical enzyme purification is presented in Table III.

Because of the low enzyme activity generally found with intact mitochondria and crude extract, it is difficult to determine the extent of purification with assurance. The presence of DNase and nucleotidases in mitochondria (32) could account for the low activities associated with these fractions. For example, the reason for the large increase in total units (4fold) observed in Step 2 of the purification procedure compared to Step 1 might result from the removal of some inhibitory nuclease; this point will be discussed in detail later in this report. Another factor which makes it difficult to assess the level of purification is the increased instability of the enzyme with purification.

Some Requirements of Partially Purified Enzyme

Requirements for the incorporation of dATP-14C by Fraction AS 50-70 are presented in Table IV. The partially purified enzyme requires Mg++, all four deoxyribonucleoside triphosphates, and an exogenous supply of DNA, requirements expected of a mitochondrial DNA polymerase.

Table IV shows that the elimination of a single deoxyribonucleoside triphosphate or all three deoxyribonucleotides reduced the extent of incorporat,ion approximately 90%. Omission of dTTP from the incubation mixture, when dATP-i4C was used as the labeled precursor, resulted in an even greater reduction of incorporation (97 ye).

The enzyme exhibited a marked dependence on Mg++ that could not completely be replaced by Mn++; the presence of both ions in the incubation mixture inhibited the activity 70 ye (Table

IV. Heat treatment of the enzyme (95” for 15 min) almost com-

pletely suppressed the activity. Pyrophosphate at a level of 10 pmoles per ml of incubation medium inhibited the incorporation

85%.

Both actinomycin D and phleomycin were equally effective in


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Issue of September 25, 1968 G. F. Kalf and J. J. Ch’ih

FIG. 1. Electron photomicrograph of washed mitochondria. (cf. “Methods”).

A five times washed mitochondrial pellet was fixed in glutaraldehyde This treatment renders the mitochondrial matrix dense and compact, with the outer mitochondrial membrane show-

ing variable degrees of separation from the inner compartment. such a preparation. The magnification is X 29,500.

Contaminating nuclei or nuclear fragments are virtually absent in

TAIILE 111

I’urijkation of enzyme from rat liver mitochondria

The standard reaction mixture for assaying the various fractions was as described in the legend to Table I. The details of the frac- tionat,ion and assay procedures are given in t’he text.

Fraction and step Ratio, Am:Axo Total activity Total protein Specific activity Yield 1 Purification

nnits* m.K unils/mg grotein % -fold

1. Crude extract.. 0.75 3,860 126 31 100 1 2. Differential ultracentrifugaticm. 0.95 14,280 60 238 368 8 3. Ammonium sulfate. 1.48 1,894 4.2 451 49 12 4. DEAE-cellulose. 1.68 818 1.2 G82 21 22

a One unit of enzyme activity is defined as that amount which will catalyze the incorporation of 1 PImole of radioact’ive deoxyribonucleotide into an acid-insoluble product. in 30 min at 37”.

causing an inhibition of enzyme activity, confirming the data previously obtained with intact mitochondria.

Dh’A resulted in an incorporation only 34y0 of that’ obtained with the native template.

Under the assay conditions generally used, the incorporation was linear with time up to 2 hours. Templale Requirements

The enzyme shows virtually a complete dependence on exogenous DS:1. Table IV presents data which show that the omission of calf thymus DS-1 from the reaction mixture caused a loss of 80 to 90’/, of the activity. The addition of DNase in the presence of template had the same effect. It is interesting that the partially purified enzyme shows a preference for native DNA. Substitution of heat-denatured calf thymus DNA for native

The preference shown by the mitochondrial enzyme for native rather than heat-denatured calf thymus DNA as template suggested that the mitochondrial enzyme might have a unique template requirement. To test this, the ability of the mitochondrial polymerase to use various DNAs was compared with that of the nuclear polymerase isolated from highly purified nuclei of the same homogenate. The AS 50-70 fraction of both


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4910 DNA Polymerase from Rat Liver Mitochondria Vol. 243, No. 18

TABLE IV TABLE V

Requirements for incorporation of W-dATP by partially puri$ed Comparison of template requirements for mitochondrial and nuclear mitochondrial DNA polymerase DNA polymerases from rat liver

The standard reaction mixture was as described in the legend The standard reaction mixture was as described in the legend to Table I, except that DNA was added at the level of 25 rg per ml of incubation mixture. Both enzymes were used as the AS

to Table I.

system

Complete system. .............................. - DNA ........................................ + Heated DNAa. ............................... + DNase (20rg). ............................. - dCTP, dGTP, and dTTP. ................ - dCTP ........................................ - dGTP ..................... .................. - dTTP., ...................................... - Mg++ ..................................... + Mn++; - Mg++, ...................... ... + Mn++ and Mg ++ (5 pmoles each) .............. + Pyrophosphate (10 pmoles) ................... + Actinomycin D (100 pg) ..................... + Phleomycin (100 pg) .................. Complete system, heateda. ......................

45 50-70 fraction

units/nzg protein

95 8

33

15 10 8

15 3 3

76

28 14 36

17 8

a The heat treatment was carried out at 95” for 15 min

enzymes was used; each showed only a slight amount of activity in the absence of added DNA. These results are presented in Table V. Enzymic activity is expressed as the percentage of incorporation of dATP-14C into acid-precipitable material by either the mitochondrial or the nuclear polymerase in the presence of 25 pg of the appropriate exogenous template. The activity was compared to that of native calf thymus DNA, which was taken as the standard and assigned a value of 100%. All values were corrected for a small amount of activity due to the presence of endogenous DNA. Relative to native calf thymus DNA, the mitochondrial polymerase did not respond very well to heat- denatured calf thymus DNA whereas the nuclear enzyme showed a 2-fold increase in activity. Another distinct difference is that the nuclear polymerase appears to utilize bacterial DNAs more efficiently than calf thymus DNA whereas the mitochondrial enzyme does not seem to be able to operate with bacterial DNA as template (Table V).

The most significant difference, however, is the behavior of the two enzymes toward mitochondrial DNA. When rat liver M-DNA* was used as the template, a 17-fold stimulation of incorporation by the mitochondrial enzyme was observed (Table

VI. The stimulation by M-DNA appears to be restricted to the

homologous M-DNA, as neither calf liver nor lamb heart M-DNA stimulated. But when rat liver M-DNA was utilized as template for the nuclear enzyme, a 4-fold stimulation was observed relative to calf thymus DNA. However, the M-DNA did not serve any more effectively as a template for the nuclear enzyme than did homologous rat liver nuclear DNA (Table V).

Presence of Contaminating Enzymes in Partially Puri$ed DNA Polymerase

The 4-fold stimulation of incorporation observed with the nuclear enzyme when M-DNA was added is puzzling. One

1 The abbreviation used is: M-DNA, mitochondrial deoxyribonucleic acid.

5@70 fraction.

Template

Calf thymus DNA Native...........................

Heated.......................... Rat liver mitochondrial DNA.. Calf liver mitochondrial DNA.

Lamb heart mitochondrial DNA. Rat liver nuclear DNA. E. coli DNA..

Mitochondrial NlKlESir polymerase polymerase

% inco*J%ralion of 1°C.dAMP’

100

34 1715

121 95 94

56

100 192

390

327 167

(1 Corrected for a small amount of endogenous activity present in the control sample (without added DNA).

TABLE VI

DNase activity in “polymerase” fractions

The details of the fractionation and assay procedures are given in the text. One unit of DNase activity is that amount of activity which causes an increase in absorbance at 260 w of 0.001

per min per ml under the given conditions at 25”.

Fraction and step Total DNase activity

units

1. Crudeextract...... .._................. 5800 2. Differential ultracentrifugation.. 775 3. Ammonium sulfate. 100 4. DEAE-cellulose. <l

possible explanation might be that the nuclear preparation contained a small amount of contaminating mitochondrial polymerase which was acting preferentially on M-DNA; conversely, the possibility also existed that the mitochondrial enzyme was contaminated with nuclear enzyme. Furthermore, as was stated above, the increase in total enzyme units from Step 1 to Step 2 in the purification procedure (Table III) indicated the possible presence of a nuclease (DNase) in the mitochondrial preparation, the removal of which would result in the retention of more acid-insoluble radioactive material.

Table VI presents data which indicate that DNase activity is present throughout the purification of the mitochondrial DNA polymerase. It was found that the crude extract contained a considerable number of units of DNase. When the crude extract was subjected to a preliminary purification by differential ultracentrifugation, the DNase units decreased 87%. After chromatography of the enzyme on DEAE-cellulose, the mitochondrial DNA polymerase contained less than 1 unit of DNase activity. Also, some terminal DNA nucleotidyl transferase appeared to be present, as evidenced by the fact that a small but significant incorporation of dATP-1% was observed to occur routinely in the absence of the three unlabeled deoxyribonucleoside triphosphates.

For these reasons, an attempt was made to purify the enzyme


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Issue of September 25, 1968 G. F. Kalf and J. J. Ch’ih 4911

of possible contaminating activities by chromatography on a TABLE VII DEAIZ-cellulose column. Separation of mitochondrial DNA polymerase from both terminal

DEAE-cellulose Chromatography addition enzyme and nuclear DNA polymerase

The standard reaction mixture for assaying the various frac- The AS 50-70 fraction was chromatographed on a column of tions was as described in the legend to Table I, except that DNA

DEAE-cellulose according to the procedure of Krakow, Coutso- was added at the level of 25 pg per ml of incubation mixture.

georgopoulos, and Canellakis (33) for the separation of the terminal addition enzyme from the nuclear DNA polymerase.

dAMP-1% incorporation by column fractions System

Elution of the column with a stepwise gradient of potassium A

phosphate at pH 7.2 gave rise to a profile which showed three ~ B I c

protein peaks (Fig. 2a). The first peak, A, came off the column ~wdes dAMP-“C incorporated/ntg protein

immediately after the void volume and generally represented Complete system”. 126 683 818

60 to 67% of the total protein applied to the column. A second - dCTP, dGTP, and

peak, B, was eluted from the column in 60 mM potassium phos- dTTPa. 228 238 528

phate, pH 7.2. Mitochondrial DNA as

This peak contained 26 to 29% of the AS 5(r70 protein. Peak C, the third fraction obtained from the column,

template.. 2760 882 Nuclear DNA as tem-

plate. 306 1261

j

a Calf thymus DNA was used as template.

contained 7 to 9% of the total protein and was eluted with 60 mM potassium phosphate, pH 7.2, plus 0.1 M ammonium sulfate.

20mM,pH 7.2 1 pH7.2 t / , The three protein peaks were individually assayed for DNA

I -I polymerase, DNase, and the terminal addition enzyme. Peak

B (a: A was found to contain virtually all of the DNase activity; less than 1 unit was present in Pea/c B (Table VI).

1.8 Each of the three peaks was assayed for DNA polymerase and 300 -

\ -I 1.4 terminal addition enzyme by testing the incorporation of dATP- ,’

:‘I ____ 1 ,-’ \ ,

‘----I\, _ 1.0 14C in the presence and in the absence of the three unlabeled

200 -- ‘\’ .\.” .--f \I 0.6 deoxyribonucleoside triphosphates.

-5

F It was shown that the incorporation of dATP-14C by the frac-

0)

,’ ‘OO-

: tions of Peaks B and C was suppressed in the absence of the *i

0 E three unlabeled precursors, but the incorporation by the frac-

.- tions of Peak A was stimulated (Table VII). These results 0

8 L hl - o-

indicate that Peak R contains the terminal addition enzyme as

(b 0 .- previously reported (33), and that Peaks B and C contain

cz polymerases. The fact that both Peak B and C contain DNA

x polymerase activity necessitated our making a distinction be-

: tween the two enzymes. On the basis of results previously 1.6 ; reported (33), it was anticipated that Peak C contained the

,’ ‘i 1.4 I :

nuclear polymerase because of the requirement for 0.1 M am-

* --------- -’ \,--,---’ ./‘.

g

I_ -I 1.2 2 monium sulfate for elution, and that Peak B perhaps contained a

1.0 distinct mitochondrial enzyme. In order to differentiate be- I

C 0.8 tween the enzymes in the two peaks, they were assayed with

200

tl I L-J&l -I A

rat liver DNA, both mitochondrial and nuclear. Table VII

B presents the results, which show that the enzyme located in

‘Ojl Peale B has a definite preference for 1CI-DNA as template, whereas the enzyme in Peak C prefers nuclear DNA. Apparently the mitochondrial DN.4 polymerase is eluted from the column

0 50 ‘00 150 200 250 in 60 mM phosphate buffer, pH 7.2, and thus is distinctly different

Fraction No. from the nuclear enzyme, which requires 0.1 M ammonium sulfate

FIG. 2. The separation of DNA polymerase on DEAE-celln- for elution. Furthermore, it is apparent that the mitochondrial

lose. A column, 1.2 X 18 cm, was prepared from DEAE-cellulose AS 50-70 fraction is contaminated with some soluble nuclear previously equilibrated with 0.02 M potassium phosphate buffer, DNA polymerase. Altogether, six DEAE-cellulose columns pH 7.2. The column was charged with 4 ml (12 mg of protein) of Fraction AS 50-70. The enzymes were eluted stepwise with 120

were run, and all of the elution patterns were found to contain

ml of 20 mM potassium phosphate, pH 7.2 (Fraction A); 80 ml of a peak, C, which represented 7 to 9% of the protein of the frac-

60 mM potassium phosphate, pH 7.2 (Fraction B) ; and 60 ml of 60 tion. Peak B represented 26 to 29% of the total protein. mM potassium phosphate, pH 7.2, plus 0.1 M ammonium sulfate (Fraction C). a, AS 50-70 fraction of mitochondrial preparation;

When purified nuclei were extracted and partially purified by

b, AS 50-70 fraction of the mixture of both mitochondria and nil- the procedure used for the mitochondrial enzyme and the AS

clei . -, protein concentration; - - - , absorbance ratio, 5&70 fraction was chromatographed on a DEAE-cellulose

A2ao:A-m. column, an elution profile similar to the one presented in Fig. 2a


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4912 DNA Polymerase from Rat Liver Mitochondria Vol. 243, No. 18

was obtained, except that Peak B now contained only 2% of the protein applied to the column whereas Peak C remained at 9% of the tot,al protein.

In order to prove unequivocally that the mitochondrial and nuclear DNA polymerases are unique enzymes, purified mitochondria and nuclei were isolated and a single extract containing both enzymes was prepared The AS 50-70 fraction was chromatographed on DEAE-cellulose; the elution pattern obtained in this experiment is presented as in Fig. 2b. The profile was similar to the one presented in Fig. 2~. Upon assay, Peak A was found to contain the terminal addition enzyme and DNase, Peak B showed DNA polymerase activity which preferentially used M-DNA, and Peak C showed polymerase activity which used nuclear DNA.

Properties of DEAE-cellulose-puri$ed Enzyme

Effect oj pH on Rate of Reaction-Maximal activity was obtained at pH 7.5 in Tris buffer. The enzymic activity was approximately 44 y. of the optimal value at pH 7.0 and 33 y0 at pH 8.0. When the reaction was carried out in potassium phosphate buffer, the curve showed an optimal pH which was shifted to the acid side to pH 7.0. In all other respects it resembled the curve obtained in Tris buffer.

Effect of Time on Incorporation Reaction-Fig. 3 presents the curve obtained when the incorporating activity of the mito-

200 -

loo-,/

:I- Tb) I v’

50

t/ I/c

04 0 20 40 60 80 100 120

Time of Incubation (min)

FIG. 3. Effect of time on the incorporation of dAMPJ4C by DEAE-cellulose-purified mitochondrial DNA polymerase. The standard reaction mixture was as described in the legend to Table I, except that in u, 5pg of M-DNA were added; in b, 1OOrg of native calf thymus DX24 were used as template. dATP-14C (30 mwmoles; specific activity, 55 mC per mmole) and 50 pg of enzyme protein were used in all incltbations. Incubation time was as shown.

75 -0 aJ 0 i 60- ;

f 45-

PI 3 30- -0

= 00 0 20 40 60 80 100 120

Enzyme Protein Concentration (pg)

FIG. 4. Linearity of the reaction with enzyme concentration. The standard reaction mixture was as described in the legend to Table I, except that IOOpg of native calf thymus DNA were used as template. Enzyme protein was added as shown.

chondrial DNA polymerase was tested with time. The reaction was linear up to 2 hours when M-DNA (5 pg) was used as template (Fig. 3~). However, when calf thymus DNA (100 pg) was used, the reaction was linear only for the first hour and then began to level off (Fig. 3b). It can be seen that the maximal incorporation of dATP-1% was much higher when M-DNA was used.

Linearity of Reaction with Enzyme Concentration-The polymerization reaction was linear with enzyme concentration up to at least 120 pg of enzyme protein when either calf thymus or M-DNA was used as template. Fig. 4 presents the data obtained with calf thymus DNA.

Divalent Metal Requirement-The purified enzyme requires added Mg+f. In the absence of MgClz virtually no activity was observed with either calf thymus or M-DNA. Under standard assay conditions, the optimal Mg++ concentration appears to be 12 X 1OW M. At 6 X lop3 M and 2.4 X 10e2 M, 18% and 16’%, respectively, of maximal activity were observed. The divalent ion requirement can be replaced only partially by Mn++.

Identification of Product-In order to establish that the dATP- 14C was incorporated into internucleotide linkage in DNA and that the labeled DNA species was indeed mitochondrial, the following experiments were carried out. A large scale incubation was set up with DEAE-cellulose-purified polymerase, M-DNA as template, and dATP-W4C as the labeled precursor. The incubation was allowed to proceed for 90 min under optimal conditions. At the end of this time, labeled M-DNA was recovered by phenol extraction.

This DNA contained 4360 dpm, for a total incorporation of 36 pprnoles of dATP-*4C. The following evidence indicated that the labeled product was DNA. (a) It gave a typical nucleic acid spectrum with a maximum absorbance at 259 rnp and a minimum at 230 mb. (b) The ratio, A2s9:AzS0, was 1.60. (c) The labeled material was resistant to alkaline hydrolysis and digestion by pancreatic RNase, whereas the radioactivity became completely solubilized upon exposure to DNase.

CsCl Gradient Centrijugafion

A preparative isopycnic centrifugation was carried out to determine the buoyant density of the newly synthesized, radio-


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1.7002

1.7002 b

-( 1

I

200

100

: u

1 ,x .- 2 ;;

200 2 2

100

3 Bouyant Density

FIG. 5. Preparative isopyncnic centrifugation of mitochondrial DNA. Samples a and b, which represent native dAMP-‘GM- DNA and renatured dAMP-‘%-M-DNA, respectively, were taken in equal volumes from the same preparation of newly synthesized M-DNA and were centrifuged simultaneously. Twenty-two fractions were obtained from each tube; they are represented by the divisions on the abscissa. Buoyant densities were determined directly from the refractive indices of the peak fractions, and are indicated over the peaks. Radioactivity of each fraction was determined by the Millipore filter disc method as described in the text. -, absorbance at 260 mp; -- -, radioactivity (counts per min).

actively labeled M-DNA relative to that of purified, unlabeled

M-DNA.

A sample of dAMP-%-DNA was mixed with highly purified

unlabeled M-DNA and centrifuged in a CsCl gradient. Ex-

amination of the absorbance of the fractions at 260 rnp indicated

that the DNA sedimented as a single homogeneous peak with a

buoyant density of 1.7002 g crnp3 (Fig. 5~). Furthermore, the

fractions containing the radioactivity were found to coincide

exactly with the peak based on optical density measurement. The ability to undergo renaturation after heat denaturation

is a distinguishing feature of M-DNA. Therefore, in or-

der to further identify the labeled product as M-DNA it was

tested for its renaturability. Labeled M-DNA was heat de-

natured and allowed to renature by slow cooling. A mixture of labeled, renatured M-DNA and unlabeled, native M-DNA was

centrifuged in a gradient of CsCl until equilibrium was attained. The gradient profile, presented in Fig. 56, shows a single peak based on radioactivity, which is coincident with the peak based on optical density measurement at 260 rnp, suggesting that the labeled product is renaturable and thus probably M-DNA.

Autocatalytic Synthesis of Mitochondrial DNA

The ability of the mitochondrial DNA polymerase to distin- guish one DNA seauence from another can be used to determine

FIG. 6. Autocatalytic synthesis of mitochondrial DNA. The 3.0-ml reaction mixture contained 300 pg of DEAE-cellulose-purified enzyme and 1 pg (initially) of M-DNA. Other components of the reaction mixture were present at a level 3 times that described in the legend to Table I, except that 127.5 mpmoles of dATPJ*C (1 PC) were used. At the indicated times, a 0.2.ml sample was removed and the radioactivity in the acid-insoluble material was determined by the Millipore filter disc method described in the text. The data are plotted against time arithmetically on the right, and semilogarithmically on the left. n , 1 pg of M-DNA as

I

template; l , control (no template added).

the degree of similarity between the synthesized product and the original M-DNA template.

One approach is to examine the kinetics of DNA synthesis at template concentrations which start below those required to saturate the enzyme. This method has been used effectively to study the autocatalytic synthesis of Q&RNA by the Q/3 replicase (34).

I f the product can serve as template, a period of autocatalytic synthesis of DNA should take place. Exponential kinetics should continue until the enzyme has been saturated with product, after which the synthesis should become linear.

To carry out this test directly, a 3-ml reaction mixture was set up which contained only 1 fig of exogenous M-DNA template and 300 pg of DEAE-cellulose-purified polymerase (300: 1 ratio of polymerase to template). The polymerization reaction was allowed to proceed for 3; hours. Samples were removed at appropriate time intervals and the extent of incorporation was determined. A control flask was run in which no exogenous M-DNA was added. The data obtained in this experiment are plotted in Fig. 6. An exponential increase in the amount of radioactively labeled DNA is evident over a period of 2 hours. The control sample (without template) showed no increase in DNA synthesis during this period, indicating the complete requirement for M-DNA in the reaction. These results indicate that the product being synthesized during the course of the reaction, under conditions of limiting template, can serve to stimulate additional polymerase molecules to activity. These results also suggest that the mitochondrial DN.4 polymerase is

10,000 .

100 . s s ’ ’ 0 80 160 240

iOO0

Time of Incubation (min)


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4914 DNA Polymerase from Rat Liver dla’tochondria Vol. 243, No. 18

able to recognize the newly synthesized product as being one which is homologous to the KI-DNA template added.

In the experiment just described there was 3.5.fold replication in a at-hour period. This was calculated in the following manner. A total of approximately 3.38 mpmoles of dAMP-i4C were incorporated into the acid-insoluble material in 33 hours (obtained by a summation of the disintegrations per min present in each aliquot and correction to a total volume of 3 ml). When this figure was multiplied by the weight of dAMP, in micro- micrograms (331), a total of 1.12 pg of dAMI’ was found to be incorporated. The micrograms of labeled DNA synthesized were determined by dividing the weight of dAMP by 0.316, the reported percentage of dAMP in rat liver WDNA (35). This calculation gave a total amount of newly synthesized AI-DNA of 3.54 kg.

DISCUSSIOI\T

These studies provide evidence for the presence of a DNA polymcrase in rat liver mitochondria. Intact mitochondria have been shown to be capable of effecting the incorporation of labeled DX.1 precursors into acid-precipitable material in vitro in a linear fashion for I hour. The incorporation is partially dependent on 1Ig++ and on all four deoxyribonucleoside triphosphates (Table IV). These results confirm the findings of others for intact mitochondria from rat liver (10, I l), yeast (la), and the mold, I’hyrasu~n polycephalum (13), which were published while our studies were in progrrss.

The hazards of contamination of mitochondrial fractions with nuclei, nuclear fragments, or both is recognized. The observed incorl)oration of deoxyribonucleotides into ILI-DN.4 does not result from this type of contamination, sinre niitochondria prepared as described do not appear to contain any contaminating iluclri by phase microscopy or nuclear fragmrnts by electron microscol)y (Fig. 1). Furthermore, the incorporation was not affected by the addition of pancreatic D?;ase to the incubation medium. However, knolvn inhibitors of l>NA polymerase such as actinomycin D and phleomycin were able to penetrate the mitocholldrial membrane and inhibit the system (Table II).

Our results with intact mitochondrin, together with those of other laboratories (10-13) strongly suggest that the observed incorporation results from the operation of an intramitochondrial DNA ~~olymerase.

Conclusive proof for the presence of such an enzyme could be obtaiiled 011ly by the isolation of the enzyme and the demonstra- tioll that it was different from the nuclear polymerase of the same tissue. This was imperative because of the inability to rule out. contamination of intact mitochondria by the soluble nuclear IjsA polynirrase. The presence of soluble nuclear polgmrrase in the supernatant fluid after homogenization of tissues in aqueous media is well known (36-39). The presence of such an enzyme bound t,o the membranes of int,act mitochondria would not be espected to contribute t,o the incorporation of labeled deosyribonucleotides by an intramitochondrial T>SA polymerase, as evidenced by the lack of DiYase inhibition of intact mitochondria, but would most certainly prove to be a source of contamination once the mitochondria were subjected to extraction.

In the work reported here, a DNA polymerase has been isolated from rat liver mitochondria and purified about 22.fold (Table III).

The AS 50-70 fraction showed all of the requirements mani-

festcd by the other known DNA polymerases in that it required Mgf+, all four deoxyribonucleosidc triphosphates, and the presence of a DNA template (Table IV). RNA could not sub- stitutc for DNA, nor was any incorporation observed in the presence of the four ribonucleoside triphosphates The incorl)oration was sensitive to DNase but not to RNase. The strong inhibition by pyrophosphate would apprar to suggest that the observed reaction is that of a polymcrase.

At the Fraction L1S 50-70 level of purification, the enzyme appeared to be virtually free of endogenous l>SA as evidenced by an .1280:11260 ratio of 1.5 and an incorl)oration rate in the absence of added DS.l of about 8% of that of the complete system. The enzyme became increasingly unstable during purification, and all attempts to prevent, loss of activity were for the most, part unsuccessful. It has been reported that glycerol was effective in preventing loss of activit:; of the polymrrase from manimalian testis (40), but, this proved ineffective with our enzyme. The instability of the enzyme might rrsult from the removal of DKA, and studies to determine this lsoiilt are in progress. Complete protection against inactivation of the E. coli polymerase was provided specifically by DNA (41).

The virtual absence of endogcnous template rnade possible an investigation of the template requirements of the mitochondrial enzyme. It was observed early in these studies that t,he enzyme showed a preference for native, double stranded DNA (Tables IV and V) . JIost purified mammalian DSX polymerases function best in the presence of heat-denatured (single stranded) polymer (42). Similar behavior is eshibitcd by the DNA polymrrase isolated from E. coli after infection with Tz or TS phage (43, 44). On the other hand, polymerases purified from certain normal bacteria (41, 43, 45) can efficiently utilize either double or single stranded template.

At least one mammalian polymerase preparation has been reported to use native DSA template preferentially, and that is the enzyme from regenerating rat liver (46). However, it. should be pointed out that this enzyme was purified from the whole homogenate and could contain SOI~C mitochondrial poly- merasc.

This distinct difference prompted an investigation into t’he template requirements of the rnitochondrial polymerase. In the hope that the requirements would prove to bc different from those of the rat liver nuclrar polymcrase, each template was compared with both enzymes. Because of t,he difficulty of obtaining RI-DXA in large quantities, calf thymus DNA was routinely used as the template, and therefore calf thymus DNA was taken as the standard and assigned a value of 100%;. The resuhs, presented in Table V, establish a unique requirement on the part of the mitochondrial enzyme for XDNA. Furthermore, the stimulation observed when XDX,l was used as t,emplate by the mitochondrial polymerase was restricted to the hornol- ogous WDNA (Table V). Rat liver nuclear DNA did not function any more effectively than did native calf thymus DNA for the mitochondrial enzyme, but worked 3 to 4 times better than calf thymus DNA with the nuclear enzyme (Table V).

Mitochondrial DNA, like bacterial DNA, is circular (47-52), and for this reason it was thought that the ability of the M-DNA to function more effectively than mammalian DNAs was due to the circularity of the molecule. That this may not be the case is indicated by the fact that E. coli DNA was a poor template for the mitochondrial enzyme, whereas it worked quite well with the nuclear enzyme (Table V). These results should be con-


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sideretl with caution, because the circularity of the E. coli DXA was not established by electron tnicroscopy before use.

It is of interest that rat liver M-DNA caused a 4-fold stimulation in the activity of the nuclear enzyme relative to calf thymus DSA but not relative to homologous rat liver nuclear LS.4 (Table V). A l)ossible explanation for these puzzling rrsults might bc that the nuclear preparation contains a small amount of collturninnting mitochondrial polymerase which very cffec%ivcly utilizes the 11.1)NA.

As stated I)rrviously, the distinct possibility esisted that the mitochontlrial preparation was also contaminated with soluble nuclear polymcrase. Other experiments had suggested that DNase (Table VI) and terminal addition enzyme (Table VII) were also prrscnt in the AS 50-70 fraction.

(‘hroliratogr:tl,h?- of the AS 50-70 fraction on a column of DE-U%ccllulosc not only seljarated the DNase and the terminal addition enzyme as Peak A (Fig. 2~) (Table VII), but also confirmed the I)rcsence of a nuclear DNA polymerase (Peak C) that showed c>lutioll properties distinctly different from those of the n~itocholltlrial rllzyme (Peak B in Fig. 2~).

Peak B was elutcd with 60 mM phosphate buffer, pH 7.2, conditions ulrtlrr which the nuclear enzyme is not eluted, whereas Peak C colltailled material eluted under conditions known to sl)ec*ifically clutc the nuclear enzyme. In a series of six DEAE- cellulose ~~lurnns, the elution pattern showed a l)eak, C, eluted specifically with 60 11lM potassium phosphate, pH 7.2, l)lus 0.1 M

ammonium sulfate. This peak represented approximately 7 to 9yc of tht> Fraction AS 5(fiO protein.

It is of intcrtst that when purified nuclei were extracted and thr ]~)lymerus;c~ was ljurified by the procedure used for the mitoc*hondrial enzyme, the DEAE-cellulose column elution l)rofilc> was ident ical with the one presented in Fig. 2a, except that Peak B now rontained only 2% of the total protein applied to tht, column whcrcas Peak C contained the nuclear enzyme but only in the amount, (7 to 9yL) generally observed to contaminate the nlitochontlrial preparation. These results also indicated that thr nuclear cnzymc was contaminated with the mitochondrial polym~~rase, and served to confirm the esplanation offrrctl for the l-fold increase in activity of the nuclear AS 5&70 frartioll whcll >I-l)iY\;.1 was used as template (Table V). It can be seen ill Table VII that the nuclear polymerase (Peak C) from the l)E-UX c~olumn did not use XI-DSA any better than calf thymus 1)X.1, further implicating the mitorhondrial enzyme in the results obscrvrtl in Table V.

As phase and rlrctron microscopy had indicated a lack of cotitalnill:~tiolI of the mitochondrial fraction with nuclei and nucl(lar fragments, and because DNase did not inhibit inc*orpora- tioll by intact rnitochondria, the conclusion can be reached that the nuclear polymrrase must have become bound to the mitochondrial surface from the supernatant fluid in a relatively tcm~)latc-free form or in a form where DNA was inaccessible to digestion by DSasr. It is apparent that caution should be observed when studies are carried out with mitochondrial es- tracts, bcrause t.hc absence of nuclear material by phase or electron microscol)y is not a final criterion for determining the abs;ctlc*e of the nuclear enzyme itself.

The l)EAE-purified enzyme is free of the following enzyme activities: nuclear 1)XA polymerase, RNA polymcrase, terminal addition enzyme, and DNase.

dt this level of purification, the enzyme shows the same requirements as the AS 50-70 fraction. The reaction is dependent

upon an optimal Mg++ concentration of 12 m&f, and is linear with time (Fig. 3a) and amount of enzyme protein (Fig. 4) at pH 7.5.

The mitochondrial I>NA polymerase appears to have a molecular weight similar to that of the E. coli enzyme (53) by virtue of the fact that when it was chromatographed on a Sephadex G-100 column it emerged from the column immediately after the void volume; the molecular wright reported for the E. coli polymerase is apl)rosimately 100,000 (53).

It was established that the newly synthesized, labeled l)roduct was 7)X*4 by the facts that it had the same buoyant density as highly purified unlabeled 11.DSX in CsU (Fig. 5a) and that when the radioactively labeled I)Sh was heat denatured and then renatured, the radioactivity remained in the renatured sample (Fig 5b); renaturation is a distinctive property of XDiYA (47, 50).

In the light of these findings, mitochondria frotn rat liver appear to contain not only a unique DNA but also a unique DNA polymerase. It seems certain that the enzyme uses Rf-DNA as template, as judged by the autocatalytic synthesis of DSA (Fig. 6) with the use of a template concentration below that required to saturate the enzyme. This strongly indicates that the product, being synthesized can itself serve as template. Furthermore, under these conditions, a 3.5.fold increase in the hl-DKA content was observed.

The demonstration of a mitochondrial DiXh polymerase which selectively utilizes 11.DXA as template strengthens the view that mitochondrial DT\r’A synthesis is a self-contained process.

Acknc&edg~~/enf-Vc wish to acknowledge the excellent technical assistance of Mary Lou Goldkamp.

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George F. Kalf and John J. Ch'ihLiver Mitochondria

Purification and Properties of Deoxyribonucleic Acid Polymerase from Rat

1968, 243:4904-4916.J. Biol. Chem.

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Purification and Properties of Deoxyribonucleic Acid Polymerase … · 2003-02-01 · 4906 DNA Polymerase from Rat Liver Mitochondria Vol. 243, No. 1s DNA was quantitatively determined