[CANCER RESEARCH 33, 987-992,
May 1973]
Partial Characterization of the DNA-dependent DNA
Polymerases 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, and
Department of Biochemistry, Howard University College of Medicine, Washington, D. C. 20001 [H. P. M.]
SUMMARY
Four DNA-dependent DNA polymerases, which include a
soluble nuclear polymerase, the mitochondrial polymerase,
and two polymerases associated with the smooth membrane
fraction, have been partially purified from normal rat liver and
a fast-growing Morris hepatoma, 1111, by previously published
procedures. The membrane-associated
enzymes have been
designated as the 0.10 and 0.25 smooth membrane polym
erases on the basis of their elution from a diethylaminoethyl
cellulose column with the respective concentration of KC1.
The polymerases were shown to be separate enzymes by
their ability to use synthetic polymers as templates and their
response to several inhibitors. The mitochondrial
DNA
polymerase was readily distinguished from the smooth
membrane polymerases by its ability efficiently to utilize
poly(dA)d(pT)io
as template and by its complete inhibition
by concentrations of ethidium bromide which do not inhibit
the other enzymes. Furthermore, the mitochondrial enzyme is
the only one of the four polymerases which is insensitive to
inhibition by p-hydroxymercuribenzoate.
The various polymerases were also distinguished by the
effects 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 membrane
polymerases but is inhibitory to the nuclear and 0.25 smooth
membrane enzymes. Camptothecin inhibits the nuclear and
0.25 smooth membrane polymerases, stimulates the 0.10
smooth membrane enzyme, and has no effect on the
mitochondrial polymerase.
INTRODUCTION
The postmicrosomal supernatant fluid of both normal rat
liver and hepatoma contains several DNA-dependent DNA
polymerases (see Refs. 3 and 4 for complete bibliography),
which differ from one another in their preference for DNA as
a template. One such enzyme prefers native DNA and is of low
molecular weight. This polymerase has been shown to be
associated with the free ribosome fraction (2) and appears to
'This research was supported in part by Grants DRG-1086 from the
Damon Runyon Memorial Fund, CA 12714-01 to G. F. K., and
GRS-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 highly
purified rat liver nuclei (2). Associated with the smooth
membrane fraction of both normal liver and hepatoma is a
DNA polymerase of high molecular weight which prefers a
denatured DNA template; the activity of this enzyme is
considerably higher in hepatoma (3).
Another enzyme of rat liver cytoplasm, the mitochondrial
DNA polymerase, has also been characterized by a preference
for denatured DNA as a primer (16, 18). This fact suggested
that the smooth membrane and the mitochondrial polymerase
might, in fact, be identical and that an extensive comparison
of the 2 enzymes might be warranted.
While our studies were in progress, Baril et al. (2) reported
the purification of 2 enzymes associated with the smooth
membrane fraction of normal rat liver. In the course of
purification of these enzymes, 1 eluted from a DEAE-cellulose
column with 0.1 M KG, whereas the other required 0.25 M
KC1, and it was concluded that the enzyme eluting at 0.1 M
KC1 most probably represented the mitochondrial
DNA
polymerase present as contamination in the smooth membrane
fraction, but that the polymerase eluting at 0.25 M KC1 was a
distinct cytoplasmic enzyme.
We considered the possibility that the polymerase activity
associated with the smooth membrane fraction represented the
mitochondrial
DNA polymerase, recently synthesized on
cytoplasmic ribosomes and packaged in smooth membrane
vesicles for transport into the organelle. This seemed feasible,
as rat liver mitochondrial DNA polymerase is known to be
synthesized 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 attached
to microsomal membranes (14).
We report here that a comparison of the various purified
DNA-dependent
DNA polymerases present in the post
microsomal supernatant fluid of normal rat liver and hepatoma
7777 has indicated that these DNA-dependent DNA polym
erases of rat liver appear to be distinct enzymes.
MATERIALS AND METHODS
Animals and Tissues. Male Wistar rats, 175 to 225 g, were
fasted overnight before decapitation. Morris hepatoma 1111, a
poorly differentiated, fast-growing hepatocellular carcinoma
with small areas intermediate between well and poorly
differentiated
(Generations 69 to 86), was transplanted
bilaterally i.m. into female Buffalo rats at Howard University,
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987
G. R. Hunter, G. F. Kalf, and H. P. Morris
Washington, D. C., and the animals were shipped to
Philadelphia by Air Express. They were given food and water
ad libitum and the tumors were excised 2 weeks after
inoculation, at which time very little necrosis was observed.
Tissue Fractionation. A 20% homogenate of either normal
liver or hepatoma was prepared in a solution of 0.34 M
sucrose, 2 mM Tris-HCl, pH 7.0 (23°),1 mM EDTA (isolation
buffer). Nuclei and mitochondria were recovered by successive
centrifugation for 10 min at 1000 and 8500 X g.
The cell debris-nuclear pellet was extracted with 5 volumes
of isolation buffer and the suspension was centrifuged at
1,000 X g to recover any trapped mitochondria. Microsomes
were removed from the postmitochondrial supernatant fluid
by centrifugation for 90 min at 105,000 X g. The postmicrosomal supernatant fluid was then centrifuged for 15 hr at
105,000 Xg to obtain the smooth membrane fraction desig
nated as P-4 by Baril et al. (2). Free ribosomes were separated
from the smooth membranes on a sucrose gradient composed
of 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 et
al. (2).
Rat liver nuclei were obtained by treatment of the crude
nuclear fraction according to the method of Whittle et al. (21)
and mitochondria were prepared and washed 5 times as
routinely carried out in our laboratory (16). The pellets were
stored at -76° until used.
Assay of Polymerases. Unless otherwise indicated the
standard assay contained the following reagents in a final
volume of 125 jul: Tris-HCl, pH 8.0, 25 mM; magnesium
acetate, 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 Ì),100 /ug/ml; and 2 to 20 jug of the
appropriate enzyme protein. Incubation was carried out, in
duplicate, in 12-x75-mm
disposable culture tubes. After
incubation at 37°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 beaker
containing an ice-cold solution of 10% trichloroacetic acid
with 1% sodium pyrophosphate and allowed to remain for 1 hr
at 4°.The solution was then changed to 5% trichloroacetic
acid:l% pyrophosphate and the discs were allowed to remain
in it overnight. The discs were then washed successively for 15
min in ether:ethanol (1:1) and ether. They were dried and
placed in a counting vial containing 0.5 ml of Protosol in order
to elute the labeled polynucleotide adsorbed to the paper disc.
This step is essential to avoid differences in the counting
efficiency of 3H-labeled polynucleotides of different molecu
lar sizes. Such differences in counting efficiency result from a
differential penetration of the biopolymer into the bound
water 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
Purification of DNA Polymerases. Nuclear polymerase was all of the labeled polynucleotide and, under these conditions,
purified from liver by the method of Baril et al. (2). counting efficiency is the same for all of the various templates
Purification was carried through the DEAE-chromatography
used. After 10 min of exposure to Protosol, a toluene
PPO-POPOP counting mixture was added and determination of
stage, at which point the nuclear polymerase can be separated
from smooth membrane polymerases by its inability to adsorb radioactivity was carried out with a counting efficiency of
to the ion-exchange column. The breakthrough fractions
25%. Quenching was corrected for by the channel ratio
containing polymerase activity were combined, dialyzed at 4°
method with an external standard. All determinations
of
overnight against Buffer A, and stored at -76°.
radioactivity were carried out to an accuracy equal to 1% S.D.
Chemical Compounds. Unlabeled deoxynucleoside triphosThe procedure of Baril et al. (2) was also used to purify the
phates, calf thymus DNA, DNase 1 (EC 3.1.4.5), and
DNA polymerases from smooth membranes. Smooth mem
dithiothreitol were purchased from Sigma Chemical Co., St.
branes were separated from any ribosomes by sucrose gradient
centrifugation and were extracted and fractionated with an Louis, Mo. TTP-methyl-3 H (4 Ci/mmole) was obtained from
ammonium sulfate solution; the precipitate obtained at 25 to New England Nuclear, Boston, Mass. P-L Biochemicals, Inc.,
40% ammonium sulfate was dissolved in a minimal volume of Milwaukee, Wis., was the source of the synthetic templates
Buffer A and dialyzed against Buffer A. A DEAE-cellulose
poly(dA)d(pT), o, poly(rA)d(PT)10, and poly(rA)(dT). Spe
column was charged with the dialyzed preparation and the cial enzyme grade ammonium sulfate was purchased from
column was eluted successively with 0.10 and 0.25 M K.C1in Schwarz/Mann, Orangeburg, N.Y. DEAE-cellulose (Whatman
Buffer A. The tubes containing polymerase activity which DE-52) was obtained from H. Reeve Angel Co., Clifton, N. J.,
eluted at 0.10 and at 0.25 M KC1 were pooled separately and and ethidium bromide from Calbiochem, San Diego, Calif. The
the eluates were dialyzed against Buffer A and then stored at sodium salt of camptothecin was generously supplied by Dr.
—¿76°.
These 2 activities will be referred to henceforth as 0.10
Harry B. Woods of the Cancer Chemotherapy Branch of NIH.
and 0.25 smooth membrane polymerases. Our preparations of The drug was soluble to 10 mg/ml in 0.10 M Tris-HCl, pH 8.5.
these enzymes have the same properties as those reported by
Baril et al. (2) for their enzymes at the equivalent level of
RESULTS
purification.
Mitochondrial
DNA polymerase was prepared by the
Four DNA-dependent DNA polymerases which include a
method of Meyer and Simpson (19) and was purified through
soluble nuclear polymerase, the mitochondrial polymerase,
the DEAE-cellulose chromatography step. The 2nd fraction
and 2 polymerases associated with the smooth membrane
eluted stepwise from the column with 0.15 M NaCl contained
the polymerase activity. This fraction is referred to as Mt-III fraction have been partially purified from normal rat liver and
and has the same properties as previously reported for the rat a fast growing Morris hepatoma, 7777, by DEAE-cellulose
liver enzyme (19). Mt-III was stored under liquid nitrogen.
chromatography.
At this stage in their purification the
988
CANCER RESEARCH
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DNA Polymerases from Rat Liver and Hepatoma
enzymes show properties identical to those previously re
ported (2, 19) and appear to be different enzymes by their
ability to use synthetic polymers as templates and their
response to several inhibitors.
Template Requirements.
The ability of these partially
purified DNA polymerases from liver and hepatoma to use
various forms of DNA and synthetic polymers as templates is
presented in Tables 1 and 2. DNA, activated by brief
treatment with pancreatic DNase I (1), was used as a control in
each case and the activity of each enzyme with this template
has been designated as 100%. All of the polymerases showed
an absolute dependence on the presence of a template as
indicated by the complete loss of activity in the absence of
exogenous DNA and by the addition of DNase to the
incubation medium (Table 1). Native DNA did not function as
a template with any of the enzymes, thus further supporting
the contention that the enzymes are free of contaminating
nuclease activity. Denatured DNA served as a template for
purified mitochondrial DNA polymerase and to a lesser extent
for the 0.25 smooth membrane enzyme (Table 1); however,
the ability of these polymerases to use denatured DNA
decreased with increasing purification of the enzyme to the
point where activity with this template could no longer be
used as a distinguishing characteristic.
The mitochondrial and 0.25 smooth membrane enzymes
from both normal liver and hepatoma showed an absolute
requirement for the presence of all 4 deoxynucleoside
triphosphates for activity, whereas the 0.10 smooth membrane
polymerase and the nuclear polymerase consistently showed
significant activity in the absence of the 3 unlabeled
deoxynucleoside
triphosphates (Table 1). A high rate of
incorporation with fewer than 4 deoxyribonucleoside triphos
phates has been observed with certain other mammalian DNA
polymerases (2, 11,13).
Synthetic Templates. It is apparent from the data presented
in Table 2 that the mitochondrial DNA polymerase of liver
and hepatoma can very efficiently use poly(dA)d(pT)i0
as a
template when compared to a control incubation employing
activated DNA as template. Furthermore, the mitochondrial
polymerase can readily be distinguished from both of the
smooth membrane polymerases on the basis of activity with
this template (Table 2). The polymer, poly(rA)d(pT))0, is not
utilized as a template by either of the smooth membrane
polymerases or the mitochondrial polymerase from liver. The
mitochondrial polymerase from hepatoma, however, shows a
3-fold stimulation of activity relative to activated DNA when
poly(rA)d(pT), o is the template. Poly(rAXdT) did not
function as a template to any significant degree with any of
the enzymes (Table 2).
Inhibition by Ethidium Bromide. The effect of ethidium
Table 1
Template 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 were
substituted at 100 Mg/ml.The assay of polymerase was carried out as described in "Materials and Methods."
control)Mitochondrial
membraneConditions
DNA polymerase (% of
0.10 smooth membrane
NormalActivated
(normal)100(964)
Normal
Normal100(551)
33)aNative DNA
100 ( 1
<1Denatured
DNA
30Minus DNA
<1Activated
DNA
7(5
DNA plus DNase
Mg/ml)Activated
<1unlabeled
DNA minus 3
deoxynucleosidetriphosphatesHepatoma
0.25 smooth
100(219)<1 100(126)
5<1
10
3<1
20
2<1
<1
<140
<1
100(39)9
<139
<1<1
<1<1
<14
Hepatoma
100(382)
<1
<1
<1
<1
<1
40Hepatoma
30
a Nos. in parentheses, pmoles of TTP-3H incorporated per mg of protein in the control incubation.
Table 2
Synthetic 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 is
expressed as 100%. Synthetic polymers were added at a concentration of 10 Mg/ml and polymerase activity was assayed as
described in "Materials and Methods."
DNA polymerase (% change)
Mitochondrial
Polymer
Normal
Hepatoma
0.10 smooth membrane
Normal
Hepatoma
0.25 smooth membrane
Normal
Hepatoma
poly(dA)d(pT),
„¿poly(rA)d(pT)1
„¿poly(rAXdT)+3657-87-100+2415+300-88-61-98-100-61-98-100+418-6-54-21-100-100
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989
G. R. Hunter, G. F. Kalf, and H. P. Morris
A. Normal
5
10
15
B. Hepatoma
20
12345
EthidiumBromideConcentration'Ug/mD
Chart I. The effect of ethidium bromide on the activity of the
DNA-dependent DNA polymerases of rat liver and hepatoma. The
polymerase assay was performed as described in "Materials and
Methods." Ethidium bromide was added at the concentration indicated.
•¿,
0.10 smooth membrane polymerase; °,0.25 smooth membrane
polymerase; D, mitochondrial DNA polymerase; •¿,
liver nuclear DNA
polymerase.
bromide on the activity of the various polymerases is shown in
Chart 1. Levels of ethidium bromide as low as 2 Mg/ml almost
completely inhibited the mitochondrial polymerase from both
liver and hepatoma. In contrast, this concentration of the dye
slightly stimulated the activity of 0.10 smooth membrane
polymerase from both liver and hepatoma. The activity of
both the 0.25 smooth membrane and the nuclear enzyme from
liver was slightly inhibited by ethidium bromide at 2 Mg/ml
(Chart L4); however, the activity of the 0.25 smooth
membrane enzyme from hepatoma showed a slight stimulation
(Chart IB). All of the polymerases were inhibited by
concentrations of ethidium bromide greater than 10 ¿¡g.
Effect of Sodium Chloride. The effect of increasing NaCl
concentration on the activity of the polymerases is presented
in Chart 2. It can be seen that 50 mM NaCl stimulates the liver
mitochondrial polymerase 65% (Chart Z4); the enzyme from
hepatoma is optimally stimulated by 100 mM NaCl, and at this
salt concentration its activity is 250 times that of the enzyme
from 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 a
qualitatively similar stimulation by NaCl to that observed for
the mitochondrial enzyme. On the other hand, the 0.25
smooth membrane enzyme from both liver and hepatoma
shows a NaCl inhibition similar to that seen with the nuclear
enzyme; the addition of very low concentrations of NaCl
causes an inhibition of activity which is complete at 200 mM.
Although the mitochondrial and 0.10 polymerases are stimu
lated 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 are
inhibited 80% or more by 5 X 1CT4 M PHMB whereas the
4The abbreviation used is: PHMB, p-hydroxymercuribenzoate.
990
purified soluble nuclear polymerase from normal rat liver is
inhibited only 40%; these data confirm the previously reported
findings of Baril et al. (2). The tumor enzymes show a pattern
of inhibition similar to that of their counterparts from normal
liver (Chart 3B). In contrast, the mitochondrial enzyme from
both liver and hepatoma is insensitive to inhibition by PHMB
even at levels as high as 3 mM and thus can readily be
distinguished from the smooth membrane and nuclear polym
erases.
Inhibition
by Camptothecin.
The alkaloid antibiotic,
camptothecin, has potent antitumor and antileukemic activity
(8, 12, 17) and has been shown to inhibit DNA synthesis in
HeLa and L 51784 cells in culture (5, 17). It does not appear
to affect DNA synthesis by rat liver or brain mitochondria in
vitro (5). On the basis of these observations, we felt that
camptothecin might be useful for distinguishing the various
polymerases. The activity of the liver nuclear polymerase and
the 0.25 smooth membrane polymerase from liver and
hepatoma is sensitive to inhibition by the antibiotic in vitro
(Chart 4). The 0.10 smooth membrane enzyme from liver is
slightly stimulated by low levels of camptothecin, and this
stimulation is more pronounced with the hepatoma enzyme.
Mitochondrial DNA polymerase is unaffected by the drug.
DISCUSSION
The 4 DNA-dependent DNA polymerases partially purified
from rat liver and hepatoma 7777 were shown to be separate
enzymes. The mitochondrial DNA polymerase was readily
distinguished from the smooth membrane polymerases by its
ability to very efficiently use poly(dA)d(pT)|0
as template
(Table 2). The inability of the smooth membrane polymerases
from liver and hepatoma and the mitochondrial polymerase
from normal liver to use the hybrid polymers poly(rA)d(pT)i 0
and poly(rA)(dT) as effectively as activated DNA is consistent
with the known inability of cellular DNA polymerases to use
A. Normal
B. Hepatoma
80
60
40
20
o
20
40
60
80
100
0
50
100 150 200
0
50
100 150
Sodium Chloride Concentration imM)
Chart 2. The effect of NaCl on the activity of the DNA-dependent
DNA polymerases of rat liver and hepatoma. The polymerase assay was
performed as described in "Materials and Methods." »,0.10 smooth
membrane polymerase; o, 0.25 smooth membrane polymerase; °,
mitochondrial DNA polymerase; •¿,
liver nuclear DNA polymerase.
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33
DNA Polymerases from Rat Liver and Hepatoma
A. Normal
A. Normal
o
1
p-Hydroxymercuribenzoate
2
Concentration
3
B. Hepatoma
60
60
40
40
20
20
O
O
20
20
40
40
60
60
imM)
80
B. Hepatoma
O
20 40 60 80 100 O 20 40 60
CamptothecinConcentrationfug/ml)
80 100
Chart 4. The effect of camptothecin on the DNA-dependent DNA
polymerases of rat liver and hepatoma. The polymerase assay was
performed as described in "Materials and Methods." Camptothecin was
added at the concentrations indicated. »,0.10 smooth membrane
polymerase; o, 0.25 smooth membrane polymerase; a, mitochondrial
DNA polymerase; •¿,
liver nuclear DNA polymerase.
mitochondrial polymerase. No explanation is available at
present for the stimulatory effect of the drug on the 0.10
smooth membrane polymerase from liver and hepatoma.
1
2
3
Recent immunological experiments in our laboratory (G. F.
p-Hydroxymercuribenzoate
ConcentrationImM)
Kalf, G. R. Hunter, and G. Odstrchel, to be published) have
Chart 3. The effect of PHMB on the activity of the DNA-dependent
demonstrated
that antisera prepared against each of the
DNA polymerases of rat liver and hepatoma. The polymerase assay was
performed as described in "Materials and Methods" and the PHMB was purified smooth membrane polymerases and the mitochondrial
polymerase react only with the homologous polymerase
added to the concentrations indicated and in the presence of 1 HIM
antigen, thus confirming that these polymerases are separate
dithiothreitol. •¿,
0.10 smooth membrane polymerase; o, 0.25 smooth
enzymes.
membrane polymerase; °,mitochondrial DNA polymerase; •¿,
liver
On the basis of these data, neither of the smooth membrane
nuclear DNA polymerase.
polymerases appears to represent the mitochondrial DNA
these hybrid polymers as templates (11). The 3-fold increase in polymerase in the process of synthesis on cytoribosomes for
transit into the organelle while attached to smooth membrane
the activity of the mitochondrial DNA polymerase from
hepatoma with poly(rA)d(pT)i 0 as template may be the result vesicles, as we had originally thought. At the present time it is
of contamination
with RNA-dependent DNA polymerase,
not known whether any of these DNA polymerases carries out
replicative or repair synthesis.
which is known to have high activity with this polymer (10).
An activity resembling RNA-dependent DNA polymerase has
been reported to be present in rat liver and hepatoma (20) as
well as in rat liver mitochondria (6). Ethidium bromide (2 ACKNOWLEDGMENTS
Mg/ml) completely inhibits the mitochondrial polymerase but
stimulates the 0.10 smooth membrane polymerase (Chart IA),
The authors are most grateful to Karen Lowry for excellent technical
proving that these 2 activities do not represent the same assistance.
enzyme. The various polymerases can also be distinguished by
the effects of monovalent salts and the sulfhydryl poison,
PHMB, on their activities. For example, the mitochondrial
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RESEARCH
VOL.
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1973 American Association for Cancer Research.
33
Partial Characterization of the DNA-dependent DNA
Polymerases of Rat Liver and Hepatoma
George R. Hunter, George F. Kalf and Harold P. Morris
Cancer Res 1973;33:987-992.
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Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1973 American Association for Cancer Research.