partial characterization of the dna-dependent dna ...dna polymerase of high molecular weight which...

[CANCER RESEARCH 33, 987-992, May 1973]

Partial Characterization of the DNA-dependent DNAPolymerases of Rat Liver and Hepatoma1

George R. Hunter,2 George F. Kalf,3 and Harold P. Morris

Department of Biochemistry, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 fC. R. H., G. F. K.J, andDepartment of Biochemistry, Howard University College of Medicine, Washington, D. C. 20001 [H. P. M.]

SUMMARY

Four DNA-dependent DNA polymerases, which include asoluble nuclear polymerase, the mitochondrial polymerase,and two polymerases associated with the smooth membranefraction, have been partially purified from normal rat liver anda fast-growing Morris hepatoma, 1111, by previously publishedprocedures. The membrane-associated enzymes have beendesignated as the 0.10 and 0.25 smooth membrane polymerases on the basis of their elution from a diethylaminoethylcellulose column with the respective concentration of KC1.

The polymerases were shown to be separate enzymes bytheir ability to use synthetic polymers as templates and theirresponse to several inhibitors. The mitochondrial DNApolymerase was readily distinguished from the smoothmembrane polymerases by its ability efficiently to utilizepoly(dA)d(pT)io as template and by its complete inhibitionby concentrations of ethidium bromide which do not inhibitthe other enzymes. Furthermore, the mitochondrial enzyme isthe only one of the four polymerases which is insensitive toinhibition by p-hydroxymercuribenzoate.

The various polymerases were also distinguished by theeffects of monovalent salts and the alkaloid antibiotic,camptothecin, on their activities. NaCl (0.05 to 0.10 M)stimulates the mitochondrial and 0.10 smooth membranepolymerases but is inhibitory to the nuclear and 0.25 smoothmembrane enzymes. Camptothecin inhibits the nuclear and0.25 smooth membrane polymerases, stimulates the 0.10smooth membrane enzyme, and has no effect on themitochondrial polymerase.

INTRODUCTION

The postmicrosomal supernatant fluid of both normal ratliver and hepatoma contains several DNA-dependent DNApolymerases (see Refs. 3 and 4 for complete bibliography),which differ from one another in their preference for DNA asa template. One such enzyme prefers native DNA and is of lowmolecular weight. This polymerase has been shown to beassociated with the free ribosome fraction (2) and appears to

'This research was supported in part by Grants DRG-1086 from theDamon Runyon Memorial Fund, CA 12714-01 to G. F. K., andGRS-RR 5414 and CA10729 to Howard University from the NIH.

2Special Fellow of the Leukemia Society of America.3To whom correspondence should be addressed.

Received November 14, 1972; accepted January 30, 1973.

be similar to the DNA polymerase extractable from highlypurified rat liver nuclei (2). Associated with the smoothmembrane fraction of both normal liver and hepatoma is aDNA polymerase of high molecular weight which prefers adenatured DNA template; the activity of this enzyme isconsiderably higher in hepatoma (3).

Another enzyme of rat liver cytoplasm, the mitochondrialDNA polymerase, has also been characterized by a preferencefor denatured DNA as a primer (16, 18). This fact suggestedthat the smooth membrane and the mitochondrial polymerasemight, in fact, be identical and that an extensive comparisonof the 2 enzymes might be warranted.

While our studies were in progress, Baril et al. (2) reportedthe purification of 2 enzymes associated with the smoothmembrane fraction of normal rat liver. In the course ofpurification of these enzymes, 1 eluted from a DEAE-cellulosecolumn with 0.1 M KG, whereas the other required 0.25 MKC1, and it was concluded that the enzyme eluting at 0.1 MKC1 most probably represented the mitochondrial DNApolymerase present as contamination in the smooth membranefraction, but that the polymerase eluting at 0.25 M KC1 was adistinct cytoplasmic enzyme.

We considered the possibility that the polymerase activityassociated with the smooth membrane fraction represented themitochondrial DNA polymerase, recently synthesized oncytoplasmic ribosomes and packaged in smooth membranevesicles for transport into the organelle. This seemed feasible,as rat liver mitochondrial DNA polymerase is known to besynthesized on cytoplasmic ribosomes (7) and, similarly,cytochrome c is synthesized on cytoplasmic ribosomes (9, 15)and has been reported to enter the organelle only if attachedto microsomal membranes (14).

We report here that a comparison of the various purifiedDNA-dependent DNA polymerases present in the postmicrosomal supernatant fluid of normal rat liver and hepatoma7777 has indicated that these DNA-dependent DNA polymerases of rat liver appear to be distinct enzymes.

MATERIALS AND METHODS

Animals and Tissues. Male Wistar rats, 175 to 225 g, werefasted overnight before decapitation. Morris hepatoma 1111, apoorly differentiated, fast-growing hepatocellular carcinomawith small areas intermediate between well and poorlydifferentiated (Generations 69 to 86), was transplantedbilaterally i.m. into female Buffalo rats at Howard University,

MAY 1973 987

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G. R. Hunter, G. F. Kalf, and H. P. Morris

Washington, D. C., and the animals were shipped toPhiladelphia by Air Express. They were given food and waterad libitum and the tumors were excised 2 weeks afterinoculation, at which time very little necrosis was observed.

Tissue Fractionation. A 20% homogenate of either normalliver or hepatoma was prepared in a solution of 0.34 Msucrose, 2 mM Tris-HCl, pH 7.0 (23Â°),1 mM EDTA (isolation

buffer). Nuclei and mitochondria were recovered by successivecentrifugation for 10 min at 1000 and 8500 X g.

The cell debris-nuclear pellet was extracted with 5 volumesof isolation buffer and the suspension was centrifuged at1,000 X g to recover any trapped mitochondria. Microsomeswere removed from the postmitochondrial supernatant fluidby centrifugation for 90 min at 105,000 X g. The postmicro-somal supernatant fluid was then centrifuged for 15 hr at105,000 Xg to obtain the smooth membrane fraction designated as P-4 by Baril et al. (2). Free ribosomes were separatedfrom the smooth membranes on a sucrose gradient composedof 0.8 to 2.0 M sucrose in 50 mM Tris-HCl, pH 7.6 (23*); 25

mM KC1; 5 mM magnesium acetate; 1 mM dithiothreitol; 20%glycerol (Buffer A) exactly according to the method of Baril etal. (2).

Rat liver nuclei were obtained by treatment of the crudenuclear fraction according to the method of Whittle et al. (21)and mitochondria were prepared and washed 5 times asroutinely carried out in our laboratory (16). The pellets werestored at -76Â° until used.

Purification of DNA Polymerases. Nuclear polymerase waspurified from liver by the method of Baril et al. (2).Purification was carried through the DEAE-chromatographystage, at which point the nuclear polymerase can be separatedfrom smooth membrane polymerases by its inability to adsorbto the ion-exchange column. The breakthrough fractionscontaining polymerase activity were combined, dialyzed at 4Â°overnight against Buffer A, and stored at -76Â°.

The procedure of Baril et al. (2) was also used to purify theDNA polymerases from smooth membranes. Smooth membranes were separated from any ribosomes by sucrose gradientcentrifugation and were extracted and fractionated with anammonium sulfate solution; the precipitate obtained at 25 to40% ammonium sulfate was dissolved in a minimal volume ofBuffer A and dialyzed against Buffer A. A DEAE-cellulosecolumn was charged with the dialyzed preparation and thecolumn was eluted successively with 0.10 and 0.25 M K.C1inBuffer A. The tubes containing polymerase activity whicheluted at 0.10 and at 0.25 M KC1 were pooled separately andthe eluates were dialyzed against Buffer A and then stored atâ€”¿�76Â°.These 2 activities will be referred to henceforth as 0.10

and 0.25 smooth membrane polymerases. Our preparations ofthese enzymes have the same properties as those reported byBaril et al. (2) for their enzymes at the equivalent level ofpurification.

Mitochondrial DNA polymerase was prepared by themethod of Meyer and Simpson (19) and was purified throughthe DEAE-cellulose chromatography step. The 2nd fractioneluted stepwise from the column with 0.15 M NaCl containedthe polymerase activity. This fraction is referred to as Mt-IIIand has the same properties as previously reported for the ratliver enzyme (19). Mt-III was stored under liquid nitrogen.

Assay of Polymerases. Unless otherwise indicated thestandard assay contained the following reagents in a finalvolume of 125 jul: Tris-HCl, pH 8.0, 25 mM; magnesiumacetate, 10 mM; dATP, dCTP, and dGTP, 15 mM each;TTP-methyl-3H, 0.5 /jCi/1.5 nmoles; dithiothreitol, 1 mM;calf thymus DNA (native, heat denatured or "activated" by

pancreatic DNAse ÃŒ),100 /ug/ml; and 2 to 20 jug of theappropriate enzyme protein. Incubation was carried out, induplicate, in 12-x75-mm disposable culture tubes. Afterincubation at 37Â°for 60 min, a 100-jul sample of each reaction

mixture was pipetted onto a filter paper disc (Whatman No.3MM) and all of the discs were immediately placed in a beakercontaining an ice-cold solution of 10% trichloroacetic acidwith 1% sodium pyrophosphate and allowed to remain for 1 hrat 4Â°.The solution was then changed to 5% trichloroacetic

acid:l% pyrophosphate and the discs were allowed to remainin it overnight. The discs were then washed successively for 15min in ether:ethanol (1:1) and ether. They were dried andplaced in a counting vial containing 0.5 ml of Protosol in orderto elute the labeled polynucleotide adsorbed to the paper disc.This step is essential to avoid differences in the countingefficiency of 3H-labeled polynucleotides of different molecu

lar sizes. Such differences in counting efficiency result from adifferential penetration of the biopolymer into the boundwater of the paper disc and a subsequent quenching of the 3H

label in the fiber matrix of the disc.Ten min of exposure to Protosol is sufficient to solubilize

all of the labeled polynucleotide and, under these conditions,counting efficiency is the same for all of the various templatesused. After 10 min of exposure to Protosol, a toluenePPO-POPOP counting mixture was added and determination ofradioactivity was carried out with a counting efficiency of25%. Quenching was corrected for by the channel ratiomethod with an external standard. All determinations ofradioactivity were carried out to an accuracy equal to 1% S.D.

Chemical Compounds. Unlabeled deoxynucleoside triphos-phates, calf thymus DNA, DNase 1 (EC 3.1.4.5), anddithiothreitol were purchased from Sigma Chemical Co., St.Louis, Mo. TTP-methyl-3 H (4 Ci/mmole) was obtained from

New England Nuclear, Boston, Mass. P-L Biochemicals, Inc.,Milwaukee, Wis., was the source of the synthetic templatespoly(dA)d(pT), o, poly(rA)d(PT)10, and poly(rA)(dT). Special enzyme grade ammonium sulfate was purchased fromSchwarz/Mann, Orangeburg, N.Y. DEAE-cellulose (WhatmanDE-52) was obtained from H. Reeve Angel Co., Clifton, N. J.,and ethidium bromide from Calbiochem, San Diego, Calif. Thesodium salt of camptothecin was generously supplied by Dr.Harry B. Woods of the Cancer Chemotherapy Branch of NIH.The drug was soluble to 10 mg/ml in 0.10 M Tris-HCl, pH 8.5.

RESULTS

Four DNA-dependent DNA polymerases which include asoluble nuclear polymerase, the mitochondrial polymerase,and 2 polymerases associated with the smooth membranefraction have been partially purified from normal rat liver anda fast growing Morris hepatoma, 7777, by DEAE-cellulosechromatography. At this stage in their purification the

988 CANCER RESEARCH VOL. 33



enzymes show properties identical to those previously reported (2, 19) and appear to be different enzymes by theirability to use synthetic polymers as templates and theirresponse to several inhibitors.

Template Requirements. The ability of these partiallypurified DNA polymerases from liver and hepatoma to usevarious forms of DNA and synthetic polymers as templates ispresented in Tables 1 and 2. DNA, activated by brieftreatment with pancreatic DNase I (1), was used as a control ineach case and the activity of each enzyme with this templatehas been designated as 100%. All of the polymerases showedan absolute dependence on the presence of a template asindicated by the complete loss of activity in the absence ofexogenous DNA and by the addition of DNase to theincubation medium (Table 1). Native DNA did not function asa template with any of the enzymes, thus further supportingthe contention that the enzymes are free of contaminatingnuclease activity. Denatured DNA served as a template forpurified mitochondrial DNA polymerase and to a lesser extentfor the 0.25 smooth membrane enzyme (Table 1); however,the ability of these polymerases to use denatured DNAdecreased with increasing purification of the enzyme to thepoint where activity with this template could no longer beused as a distinguishing characteristic.

The mitochondrial and 0.25 smooth membrane enzymes

DNA Polymerases from Rat Liver and Hepatoma

from both normal liver and hepatoma showed an absoluterequirement for the presence of all 4 deoxynucleosidetriphosphates for activity, whereas the 0.10 smooth membranepolymerase and the nuclear polymerase consistently showedsignificant activity in the absence of the 3 unlabeleddeoxynucleoside triphosphates (Table 1). A high rate ofincorporation with fewer than 4 deoxyribonucleoside triphosphates has been observed with certain other mammalian DNApolymerases (2, 11,13).

Synthetic Templates. It is apparent from the data presentedin Table 2 that the mitochondrial DNA polymerase of liverand hepatoma can very efficiently use poly(dA)d(pT)i0 as atemplate when compared to a control incubation employingactivated DNA as template. Furthermore, the mitochondrialpolymerase can readily be distinguished from both of thesmooth membrane polymerases on the basis of activity withthis template (Table 2). The polymer, poly(rA)d(pT))0, is notutilized as a template by either of the smooth membranepolymerases or the mitochondrial polymerase from liver. Themitochondrial polymerase from hepatoma, however, shows a3-fold stimulation of activity relative to activated DNA whenpoly(rA)d(pT), o is the template. Poly(rAXdT) did notfunction as a template to any significant degree with any ofthe enzymes (Table 2).

Inhibition by Ethidium Bromide. The effect of ethidium

Table 1Template requirements for the DNA polymerases of rat liver and hepatoma

Control incubations were run under standard assay conditions with activated DNA, 100 Mg/ml. Other DNA's weresubstituted at 100 Mg/ml.The assay of polymerase was carried out as described in "Materials and Methods."

DNA polymerase (% ofcontrol)Mitochondrial

0.10 smooth membrane 0.25 smoothmembraneConditions

NormalActivated

DNA 100 ( 133)aNative

DNA<1DenaturedDNA30Minus

DNA<1ActivatedDNA plus DNase7(5

Mg/ml)ActivatedDNA minus 3<1unlabeled

deoxynucleosidetriphosphatesHepatoma

Normal100(551)

100(39)9<139<1<1<1<1

<14

40Hepatoma

Normal Hepatoma(normal)100(964)

100(126) 100(382)100(219)<110 <15<120 <13<1<1 <12<1<1 <1<140

<1 30

a Nos. in parentheses, pmoles of TTP-3H incorporated per mg of protein in the control incubation.

Table 2Synthetic polymers as templates for DNA polymerases of liver and hepatoma

The percentage of change is expressed relative to the control incubation which contained activated DNA, 100 Mg/ml,and isexpressed as 100%. Synthetic polymers were added at a concentration of 10 Mg/ml and polymerase activity was assayed asdescribed in "Materials and Methods."

DNA polymerase (% change)

Mitochondrial 0.10 smooth membrane 0.25 smooth membrane

Polymer Normal Hepatoma Normal Hepatoma Normal Hepatoma

poly(dA)d(pT),â€ž¿�poly(rA)d(pT)1â€ž¿�poly(rAXdT)+3657-87-100+2415+300-88-61-98-100-61-98-100+418-6-54-21-100-100

MAY 1973 989




A. Normal B. Hepatoma

5 10 15 20 12345EthidiumBromideConcentration'Ug/mD

Chart I. The effect of ethidium bromide on the activity of theDNA-dependent DNA polymerases of rat liver and hepatoma. Thepolymerase assay was performed as described in "Materials andMethods." Ethidium bromide was added at the concentration indicated.

â€¢¿�,0.10 smooth membrane polymerase; Â°,0.25 smooth membranepolymerase; D, mitochondrial DNA polymerase; â€¢¿�,liver nuclear DNApolymerase.

bromide on the activity of the various polymerases is shown inChart 1. Levels of ethidium bromide as low as 2 Mg/ml almostcompletely inhibited the mitochondrial polymerase from bothliver and hepatoma. In contrast, this concentration of the dyeslightly stimulated the activity of 0.10 smooth membranepolymerase from both liver and hepatoma. The activity ofboth the 0.25 smooth membrane and the nuclear enzyme fromliver was slightly inhibited by ethidium bromide at 2 Mg/ml(Chart L4); however, the activity of the 0.25 smoothmembrane enzyme from hepatoma showed a slight stimulation(Chart IB). All of the polymerases were inhibited byconcentrations of ethidium bromide greater than 10 Â¿Â¡g.

Effect of Sodium Chloride. The effect of increasing NaClconcentration on the activity of the polymerases is presentedin Chart 2. It can be seen that 50 mM NaCl stimulates the livermitochondrial polymerase 65% (Chart Z4); the enzyme fromhepatoma is optimally stimulated by 100 mM NaCl, and at thissalt concentration its activity is 250 times that of the enzymefrom normal liver (Chart 2B). The same concentration of NaCl(50 mM) that stimulates the mitochondrial enzyme 65%inhibits the rat liver nuclear polymerase 80% (Chart 2A).

The 0.10 smooth membrane enzyme from liver shows aqualitatively similar stimulation by NaCl to that observed forthe mitochondrial enzyme. On the other hand, the 0.25smooth membrane enzyme from both liver and hepatomashows a NaCl inhibition similar to that seen with the nuclearenzyme; the addition of very low concentrations of NaClcauses an inhibition of activity which is complete at 200 mM.Although the mitochondrial and 0.10 polymerases are stimulated by 50 mM NaCl, they are both inhibited by 150 mM salt.

Inhibition by PHMB.4 Data presented in Chart 3A show

that both the 0.10 and 0.25 smooth membrane enzymes areinhibited 80% or more by 5 X 1CT4 M PHMB whereas the

purified soluble nuclear polymerase from normal rat liver isinhibited only 40%; these data confirm the previously reportedfindings of Baril et al. (2). The tumor enzymes show a patternof inhibition similar to that of their counterparts from normalliver (Chart 3B). In contrast, the mitochondrial enzyme fromboth liver and hepatoma is insensitive to inhibition by PHMBeven at levels as high as 3 mM and thus can readily bedistinguished from the smooth membrane and nuclear polymerases.

Inhibition by Camptothecin. The alkaloid antibiotic,camptothecin, has potent antitumor and antileukemic activity(8, 12, 17) and has been shown to inhibit DNA synthesis inHeLa and L 51784 cells in culture (5, 17). It does not appearto affect DNA synthesis by rat liver or brain mitochondria invitro (5). On the basis of these observations, we felt thatcamptothecin might be useful for distinguishing the variouspolymerases. The activity of the liver nuclear polymerase andthe 0.25 smooth membrane polymerase from liver andhepatoma is sensitive to inhibition by the antibiotic in vitro(Chart 4). The 0.10 smooth membrane enzyme from liver isslightly stimulated by low levels of camptothecin, and thisstimulation is more pronounced with the hepatoma enzyme.Mitochondrial DNA polymerase is unaffected by the drug.

DISCUSSION

The 4 DNA-dependent DNA polymerases partially purifiedfrom rat liver and hepatoma 7777 were shown to be separateenzymes. The mitochondrial DNA polymerase was readilydistinguished from the smooth membrane polymerases by itsability to very efficiently use poly(dA)d(pT)|0 as template(Table 2). The inability of the smooth membrane polymerasesfrom liver and hepatoma and the mitochondrial polymerasefrom normal liver to use the hybrid polymers poly(rA)d(pT)i 0and poly(rA)(dT) as effectively as activated DNA is consistentwith the known inability of cellular DNA polymerases to use

A. Normal B. Hepatoma

80

60

40

20o

20

40

60

80

1000 50 100 150 200 0 50

Sodium Chloride Concentration

100 150imM)

4The abbreviation used is: PHMB, p-hydroxymercuribenzoate.

Chart 2. The effect of NaCl on the activity of the DNA-dependentDNA polymerases of rat liver and hepatoma. The polymerase assay wasperformed as described in "Materials and Methods." Â»,0.10 smooth

membrane polymerase; o, 0.25 smooth membrane polymerase; Â°,mitochondrial DNA polymerase; â€¢¿�,liver nuclear DNA polymerase.




DNA Polymerases from Rat Liver and Hepatoma

A. Normal A. Normal B. Hepatoma

1 2 3p-Hydroxymercuribenzoate Concentration imM)

B. Hepatoma

1 2 3p-HydroxymercuribenzoateConcentrationImM)

Chart 3. The effect of PHMB on the activity of the DNA-dependentDNA polymerases of rat liver and hepatoma. The polymerase assay wasperformed as described in "Materials and Methods" and the PHMB was

added to the concentrations indicated and in the presence of 1 HIMdithiothreitol. â€¢¿�,0.10 smooth membrane polymerase; o, 0.25 smoothmembrane polymerase; Â°,mitochondrial DNA polymerase; â€¢¿�,livernuclear DNA polymerase.

these hybrid polymers as templates (11). The 3-fold increase inthe activity of the mitochondrial DNA polymerase fromhepatoma with poly(rA)d(pT)i 0 as template may be the resultof contamination with RNA-dependent DNA polymerase,which is known to have high activity with this polymer (10).An activity resembling RNA-dependent DNA polymerase hasbeen reported to be present in rat liver and hepatoma (20) aswell as in rat liver mitochondria (6). Ethidium bromide (2Mg/ml) completely inhibits the mitochondrial polymerase butstimulates the 0.10 smooth membrane polymerase (Chart IA),proving that these 2 activities do not represent the sameenzyme. The various polymerases can also be distinguished bythe effects of monovalent salts and the sulfhydryl poison,PHMB, on their activities. For example, the mitochondrialenzyme is the only 1 of the polymerases which is insensitive toinhibition by PHMB (Chart 3). NaCl (0.05 to 1.0 M) stimulatesthe activity of the mitochondrial and 0.10 smooth membraneenzymes but is inhibitory to the nuclear and 0.25 smoothmembrane enzymes (Chart 2).

Finally, the various DNA polymerases can be distinguishedby the use of the antibiotic, camptothecin, which inhibits the0.25 smooth membrane and nuclear polymerases, stimulatesthe 0.10 smooth membrane enzyme, and has no effect on the

o

60

40

20

O

20

40

60

60

40

20

O

20

40

60

80

O 20 40 60 80 100 O 20 40 60 80 100CamptothecinConcentrationfug/ml)

Chart 4. The effect of camptothecin on the DNA-dependent DNApolymerases of rat liver and hepatoma. The polymerase assay wasperformed as described in "Materials and Methods." Camptothecin was

added at the concentrations indicated. Â»,0.10 smooth membranepolymerase; o, 0.25 smooth membrane polymerase; a, mitochondrialDNA polymerase; â€¢¿�,liver nuclear DNA polymerase.

mitochondrial polymerase. No explanation is available atpresent for the stimulatory effect of the drug on the 0.10smooth membrane polymerase from liver and hepatoma.

Recent immunological experiments in our laboratory (G. F.Kalf, G. R. Hunter, and G. Odstrchel, to be published) havedemonstrated that antisera prepared against each of thepurified smooth membrane polymerases and the mitochondrialpolymerase react only with the homologous polymeraseantigen, thus confirming that these polymerases are separateenzymes.

On the basis of these data, neither of the smooth membranepolymerases appears to represent the mitochondrial DNApolymerase in the process of synthesis on cytoribosomes fortransit into the organelle while attached to smooth membranevesicles, as we had originally thought. At the present time it isnot known whether any of these DNA polymerases carries outreplicative or repair synthesis.

ACKNOWLEDGMENTS

The authors are most grateful to Karen Lowry for excellent technicalassistance.

REFERENCES

1. Aposhian, H. J., and Kornberg, A. Enzymatic Synthesis ofDeoxyribonucleic Acid. IX. The Polymerase Formed after T2Bacteriophage Infection of Escherichia coli: A New Enzyme. J.Biol. Chem., 2J7.- 519-525, 1962.

2. Baril, E. F., Brown, O. E., Jenkins, M. D., and Laszlo, J.Deoxyribonucleic Acid Polymerase with Rat Liver Ribosomes andSmooth Membranes. Purification and Properties of the Enzymes.Biochemistry, 10: 1981-1992, 1971.

MAY 1973 991




3. Baril, E. F., Jenkins, M. D., Brown, O. E., and Laszlo, i. DNAPolymerase Activities Associated with Smooth Membranes andRibosomes from Rat Liver and Hepatoma Cytoplasm. Science,769. 87-89, 1970.

4. Barii, E., and Laszlo, J. Sub-cellular Localization and Characterization of DNA Polymerases from Rat Liver and Hepatomas. Advan.Enzyme Regulation, 9: 184-204, 1971.

5. Bosmann, H. B. Camptothecin Inhibits Macromolecular Synthesisin Mammalian Cells but Not in Isolated Mitochondria or E. coli.Biochem. Biophys. Res. Commun., 41: 1412-1419, 1970.

6. Bosmann, H. B. Mitochondria! Autonomy. Synthesis of DNA fromRNA templates in Isolated Mammalian Mitochondria. FederationEuropean Biochem. Soc. Letters, 19: 27-29, 1971.

7. Ch'ih, J. J., and Kalf, G. F. Studies on the Biosynthesis of the DNA

Polymerase of Rat Liver Mitochondria. Arch. Biochem. Biophys.,133: 38-45,1969.

8. Gallo, R. C., Whang-Peng, J. and Adamson, R. H. Studies on the

Antitumor Activity, Mechanism of Action and Cell Cycle Effectsof Camptothecin. J. Nati. Cancer Inst., 46: 789-795, 1971.

9. Gonzalez-Cadavid, N. F., and Campbell, P. N. The Biosynthesis ofCytochrome c. Sequence of Incorporation in vivo of '4 C-Lysine

into Cytochrome c and Total Proteins of Rat Liver SubcellularFractions. Biochem. J., 105: 443-450, 1967.

10. Goodman, N. C., and Spiegelman, S. Distinguishing ReverseTranscriptase of an RNA Tumor Virus from Other Known DNAPolymerases. Proc. Nati. Acad. Sei. U. S., 68: 2203-2206, 1971.

11. Greene, R., and Korn, D. Partial Purification and Characterizationof Deoxyribonucleic Acid Polymerase from KB Cells. J. Biol.Chem., 245: 254-261, 1970.

12. Hart, L. G., Coll, J. B., and Oliverio, J. T. A Fluorometric Method

for Deterjnination of Camptothecin in Plasma and Urine. CancerChemotherapy Rept., 53: 211-214,1969.

13. Howk, R., and Wang, T. Y. DNA Polymerase from Rat LiverChromosomal Proteins. I. Partial Purification and General Characterization. Arch. Biochem. Biophys., 133: 238-246,1969.

14. Kadenbach, B. Synthesis of Mitochondrial Proteins. The Synthesisof Cytochrome c in vitro. Biochem. Biophys. Acta, 138: 651-654,1967.

15. Kadenbach, B. Biosynthesis of Cytochrome c. The Sites ofSynthesis of Apoprotein and Holoenzyme. European J. Biochem.,12: 392-398, 1970.

16. Kalf, G. F., and Ch'ih, J. J. Purification and Properties of

Deoxyribonucleic Acid Polymerase from Rat Liver Mitochondria.J. Biol. Chem., 243: 4904-4916, 1968.

17. Kessel, D. Effects of Camptothecin on Macromolecule Synthesis byMurine Leukemia Cells. Proc. Am. Assoc. Cancer Res., 12: 4, 1971.

18. Meyer, R. R., and Simpson, M. V. DNA Biosynthesis inMitochondria: Differential Inhibition of Mitochondrial and NuclearDNA Polymerases by the Mutagenic Dyes Ethidium Bromide andAcriflavin. Biochem. Biophys. Res. Commun., 34: 238-244, 1969.

19. Meyer, R., and Simpson, M. V. Deoxyribonucleic Acid Biosynthesis in Mitochondria. Purification and General Properties of RatLiver Mitochondrial Deoxyribonucleic Acid Polymerase. J. Biol.Chem., 245: 3426-3435, 1970.

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21. Whittle, E. D., Bushnell, D. E., and Potter, J. R. RNA Associatedwith the Outer Membrane of Rat Liver Nuclei. Biochim. Biophys.Acta, 767:41-50, 1968.




1973;33:987-992. Cancer Res George R. Hunter, George F. Kalf and Harold P. Morris Polymerases of Rat Liver and HepatomaPartial Characterization of the DNA-dependent DNA

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