59
Biochimica et Biophysica Acta, 414 (1975) 59--70 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 98447
A DNA-DIRECTED DNA POLYMERASE FROM MURINE L I V E R MITOCHONDRIA
NORMAN B. HECHT
Department of Biology, Tufts University, Medford, Mass. 02155 (U.S.A.) (Received June 3rd, 1975)
Summary A DNA-directed DNA polymerase has been isolated from murine liver mitochondria. The mitochondrial DNA polymerase is distinguishable from other DNA polymerases found in the nucleus and cytosol of murine cells by several enzymatic and physical properties. It is stimulated 5--6-fold by 0.15 M KC1, does not require a sulfhydryl reducing agent for activity, and is inhibited by ethidium bromide or ATP. The enzyme has a sedimentation coefficient of 8.8 S in the presence of up to 0.5 M KC1, a molecular weight of 150--170 000, and utilizes natural templates in the following order of preference: activated DNA (100%), single stranded DNA (24%), and native DNA (5%).
Introduction In addition to containing a unique DNA, mitochondria isolated from several different sources have been shown to possess a unique DNA polymerase. This enzyme has been reported to differ from the DNA polymerases found in the nuclear and cytosol fractions of the cell by the criteria of: molecular weight, sensitivity to inhibitors, chromatographic behavior, primer-template utilization, and requirements for maximal activity [ 1--10]. Despite the general agreement for the existence of a distinct DNA polymerase in mitochondria, a comparison of some of the properties o f the enzymes isolated from yeast, rat liver, and HeLa cells suggests differences between mitochondrial DNA polymerases. For example, the molecular weight has been reported to be a b o u t 150 000 for the rat liver and yeast mitochondrial D N A polymerases while the HeLa cell enzyme has a molecular weight of 106 000 [2,9,11]. In addition, the enzymatic activity o f crude or highly purified rat liver mitochondrial DNA polymerase is dramatically stimulated by ionic strengths of 0.15 M KC1 whereas the mitochondrial enzymes isolated from HeLa cells or yeast do not show a similar response to elevated ionic strength [2,4,6,9,10]. These observations and the
60 fact that we wished to relate a DNA-directed DNA polymerase isolated from mature spermatozoa [12] to other cellular D N A polymerases p r o m p t e d us to examine the DNA polymerase activity found in purified mitochondria. After aqueous subcellular fractionation, murine liver cells contain a low molecular weight DNA-directed DNA polymerase (~) in the nuclear fraction and two distinguishable DNA-directed DNA polymerases in the cytosol [13-15]. The cytosol enzymes are distinguishable by the criteria of salt sensitivity, chromatographic properties, inhibition by N-ethylmaleimide, template preference, and size [ 1 3 - - 1 5 ] . One cytosol enzyme, dissociable by 0.25 M KC1 to an active form of DNA polymerase of sedimentation coefficient 3.5 S, shares many chemical/physical properties with the DNA polymerase ~ suggesting it may represent a polymeric form of ~ monomers. The size, response to physiological change, and reaction properties of the other cytosol DNA polymerase suggest it is the DNA polymerase ~ [ 1 5 ] . In this communication I describe the properties of a DNA-directed DNA polymerase isolated from purified mitochondria of murine liver. This enzyme differs from other murine DNA polymerases found in the nucleus or cytosol by several criteria. When compared to the mitochondrial DNA polymerases from yeast, HeLa cells, and rat liver, it most closely resembles the enzyme from rat liver. Materials and Methods
Materials [ 3H] TTP (spec. act. 40 Ci/mmol) was purchased from New England Nuclear, Boston, Mass. Schwarz-Mann, Orangeburg, N.Y. was the supplier of dATP, dGTP, dTTP, dCTP and enzyme grade sucrose. Ethidium bromide, calf thymus DNA, and fraction IV bovine serum albumin were purchased from Sigma Chemical Co., St. Louis, Missouri. Phosphocellulose (P-11) was a p r o d u c t of Whatman, Co., U.K. Sephadex G-200 was obtained from Pharmacia, Inc., Piscataway, N.J. Diethylaminoethyl cellulose (Cellex-D) was purchased from Bio Rad Laboratories, Richmond, Cal. The sodium salt of camptothecin was generously provided by Dr Harry B. Woods of the Cancer Chemotherapy Branch of N.I.H. Mice (mature male CD-1 strain) were obtained from Charles River Breeding Laboratories, Inc., Wilmington, Mass. Preparation of mi tochondria Mice were killed by cervical dislocation, the livers were removed and immediately washed in suspension buffer {0.25 M sucrose containing 0.02 M Tris, pH 8.1, 1 mM CaC12, 3 mM ~-mercaptoethanol) at 4°C. The livers were minced, washed several times more with the suspension buffer, homogenized b y hand in a Teflon and glass homogenizer (10 X), and filtered through 4 layers of cheese cloth. The nuclear fraction was removed by centrifugation of the filtrate at 650 X g for 15 min. in a Sorvall HB-4 swinging bucket rotor and the supernatant was centrifuged at 13 000 X g for 30 min. The pellet, containing mitochondria, was resuspended in suspension buffer and recentrifuged at 13 000 X g for 30 min. This recentrifugation step was repeated three times. The final pellet was resuspended and layered over a linear sucrose gradient of
61 30% to 65% sucrose (dissolved in suspension buffer) and centrifuged for 90 min. at 24 000 rev./min in a SW27 rotor in a Beckman L2-65 ultracentrifuge. The mitochondrial band was removed, washed with suspension buffer, pelleted, and either used immediately or stored in liquid nitrogen. In a typical preparation 1 g of mouse liver (wet weight) yielded approximately 2 mg of mitochondrial protein.
Preparation o f mitochondrial DNA polymerase extract Frozen or fresh mitochondria were suspended in extraction buffer (0.02 M Tris, pH 8.1, 0.05 M NaC1, 3 mM fi-mercaptoethanol, 1 mM EDTA, 20% ethylene glycol) containing 1 M KC1 and sonicated for 60 s at a setting of 4 with a Branson sonicator (Model 185E) followed by incubation at 4°C for 60 min. The extract was centrifuged at 105 000 X g for 60 min in a'Beckman type 65 rotor and the supernatant was decanted and dialyzed overnight against extraction buffer. This salt extraction procedure was effective in solubilizing over 96% of the DNA polymerase activity. DEAE-cellulose chromatography Cellex-D was prepared as previously described [13] and equilibrated with extraction buffer. The dialyzed enzyme extract containing 18 mg of protein was adsorbed onto a DEAE-cellulose column at a proportion of 1 mg of protein/g of Cellex-D. The column was washed with several column volumes of the same buffer and eluted with a linear gradient from 0.05 M to 0.55 M NaC1 containing 0.02 M Tris, pH 8.1, 3 mM fi-mercaptoethanol, 1 mM EDTA, and 20% ethylene glycol. Fractions (0.7 ml) were collected directly into bovine serum' albumin (1 mg/ml) and aliquots (0.02 ml) were assayed for DNA polymerase activity. Phosphocellulose chromatography o f the mitochondrial extract Phosphocellulose was washed by the method of Burgess [ 16]. The pooled peak fractions of the DNA polymerase obtained from the DEAE-cellulose column (see Results) were dialyzed against extraction buffer and adsorbed onto phosphocellulose columns (1 X 15 cm) that had been equilibrated with extraction buffer. The columns were washed with several bed volumes of this buffer and then eluted with linear gradients from 0 to 1.0 M KC! in extraction buffer. Fractions (0.75 ml) were collected directly into bovine serum albumin (1 mg/ml final concentration) and aliquots (0.02 ml) were assayed for DNA polymerase activity. Gel filtration o f mitochondrial DNA polymerase An aliquot (1 ml) of th~ pooled peak tubes obtained from phosphocellulose chromatography (tubes 15--18 of Fig. 1) was dialyzed and layered on a Sephadex C~200 column (0.8 X 50 cm) equilibrated with extraction buffer containing 0.25 M KC1. The exclusion volume (V0) was determined by blue dextran marker and the column was calibrated with catalase, lactic dehydrogenase, alkaline phosphatase, ovalbumin, and cytochrome c.
62 5o! J 40
'o
~0
o
_~ 2o rzl
5
I0 15 FRACTION NUMBER
20
25
Fig. 1. P h o s p h o e e n u l o s e c h r o m a t o g r a p h y o f an e x t r a c t o f m u r i n e l i v e r m i t o c h o n d r i a . T h e f r a c t i o n s c o n r a i n i n g t h e D N A p o l y m e r a s e p e a k o b t a i n e d b y D E A E - c e l l u l o s e c h r o m a t o g r a p h y a n d c o n t a i n i n g 14 m g of p r o t e i n w e r e p o o l e d , d i a l y z e d a n d c h r o m a t o g r a p h e d o n a 1 × 15 c m p h o s p h o c e l l u l o s e c o l u m n . E l u t i o n w a s c a r r i e d o u t as d e s c r i b e d u n d e r M a t e r i a l s a n d M e t h o d s a n d a l i q u o t s ( 0 . 0 2 m l ) w e r e a s s a y e d f o r D N A p o l y m e r a s e as described.
Sucrose gradient cen trifuga tion Sedimentation coefficients were determined using the m e t h o d of Martin and Ames [17]. A dialyzed aliquot (0.2 ml} from the peak tubes of the phosphocellulose column was layered over preformed 5 to 20% sucrose gradients (sucrose dissolved in extraction buffer or extraction buffer + 0.5 M KC1) and centrifuged for 16 h at 55 000 rev./min at 4°C in a SW 65 rotor in a Beckman L2-65. Aliquots (0.01 ml) of alkaline phosphatase (S20,w = 6.3) and catalase ($20,w = 11.3) were added to the DNA polymerase and run as marker proteins. Assay conditions for mitochondrial D N A polymerase The standard reaction mixture (0.3 ml) contained 5 pmol Tris • HC1 buffer at pH 8.8, 2.5 pmol MgC12, 3 pmol fl-mercaptoethanol, 40 pmol KC1, 160 nmol activated calf t h y m u s DNA, 10 nmol each of dCTP, dGTP, dATP, 1 nmol of [ZH]TTP (100--750 cpm/pmol) and enzyme. Incubation was for 60 min at 37 °C. The calf t h y m u s DNA was activated by the m e t h o d of Aposhian and Kornberg [18]. After incubation, the assay tubes were placed in ice and the reaction was stopped by the addition of 0.5 ml cold 1 M HC104 containing 0.02 M sodium pyrophosphate. The precipitates were collected on glass fiber filters (Whatman GF/A) and washed with 10 ml of 5% HC104 containing 0.02 M sodium pyrophosphate and with 10 ml of 95% ethanol. The filters were dried and the bound radioactivity was counted in a liquid scintillation counter. All assays were performed in duplicate.
63 Results
Partial purification of DNA polymerase from murine liver mitochondria The procedure for extracting the murine mitochondria described under Materials and Methods solubilized over 96% of the total DNA-directed DNA polymerase but only released 20--25% of the total mitochondrial protein. The combination of high ionic strength (1 M KC1) and sonication seems essential for maximal release of the DNA polymerase since sonication at lower ionic strengths (extraction buffer in the absence of 1 M KC1) gave variable results, often releasing less than 50% of the total DNA polymerase activity from the mitochondrial pellet. Extraction of the mitochondria at 1 M KC1 in the absence o f sonication allowed 20--40% of the enzyme to remain in the pellet fraction. After dialysis, the mitochondrial extract was chromatographed on a DEAE-cellulose column. Under the conditions used, the DNA polymerase b o u n d quantitatively and upon application of a salt gradient eluted at 0.24 M NaC1. These results are identical to those we previously reported for a mitochondrial DNA polymerase from mouse testis [13]. Recoveries of enzyme activity ranged between 88 and 106% o f the activity from the initial mitochondrial extract. It was essential to collect the DNA polymerase eluted from either DEAE-cellulose or phosphocellulose columns directly into bovine serum albu20
1
A
%
IO
,6
8
5
s
o_
~
2o
z_
Z Io
5
I0 15 VOLUME (ML)
20
25
5
IO 15 FRACTION NUMBER
20
25
Fig. 2. Gel f i l t r a t i o n of d i a l y z e d f r a c t i o n s f r o m a m i t o c h o n d r i a l liver p h o s p h o c e l l u l o s c c o l u m n . T h e fractions containing the peak of DNA polymerase activity from phosphocelhdose columns were pooled, d i a l y z e d , a n d 1.0 m l was l a y e r e d o v e r a 0.8 X 50 c m S e p h a d e x G - 2 0 0 c o l u m n t h a t w a s e q u i l i b r a t e d w i t h e x t r a c t i o n b u f f e r c o n t a i n i n g 0 . 2 5 M KCI. F r a c t i o n ( 0 . 5 m l ) w e r e c o l l e c t e d a n d 0 . 0 5 m l s a m p l e s w e r e a s s a y e d f o r D N A p o l y m e r a s e . T h e c o l u m n w a s c a l i b r a t e d as d e s c r i b e d in Materials and M e t h o d s . R o u t i n e ly 4 0 - - 6 0 % o f t h e l o a d e d D N A p o l y m e r a s c was r e c o v e r e d . Fig. 3. S u c r o s e d e n s i t y g r a d i e n t cellulose c o l u m n . A l i q u o t s (0.2 layered over p r e f o r m e d sucrose t i o n b u f f e r ; B, s u c r o s e dissolved
c e n t r i f u g a t i o n o f d i a l y z e d f r a c t i o n s f r o m a m i t o c h o n d r i a l liver p h o s p h o ml) from the peak tubes o f a phosphocellulose c o l u m n were dialyzed and g r a d i e n t s . S e d i m e n t a t i o n w a s to t h e right. A, s u c r o s e dissolved in e x t r a c in e x t r a c t i o n b u f f e r c o n t a i n i n g 0.5 M KC1.
64 min to maintain good recoveries (greater than 85%) o f the loaded DNA polymerase. If bovine serum albumin was omitted, a maximum recovery of 50% was obtained. This has made it difficult to accurately calculate specific activities during the partial purification stages b u t has ensured that the enzyme fraction that was eluted from phosphocellulose represents between 80 and 90% o f the total activity from the initial mitochondrial extract. The reason that bovine serum albumin serves to increase the recovery of the mitochondrial D N A polymerase is not clear. However, it may help to reduce the "stickiness" o f the DNA polymerase since this enzyme tends to stick to glass quite readily and also aggregates in low ionic strength extraction buffers (Fig. 3A). The peak tubes elu-ting at 0.24 M NaC1 from the DEAE-cellulose were combined, dialyzed and further purified by phosphocellulose chromatography (Fig. 1). The DNA polymerase activity bound quantitatively and eluted as a sharp peak at 0.56 M KC1. The recovery of the DNA polymerase from the phosphocellulose column was nearly quantitative representing a recovery of 81--92% of the DNA polymerase from the mitochondrial extract. The fractions containing enzyme were combined, dialyzed, and stored under liquid nitrogen. All further investigations concerning the requirements and properties of the murine mitochondrial DNA polymerase were carried o u t using pooled peak fractions derived from phosphocellulose columns.
Requirements of mitochondrial DNA polymerase The requirements of the partially purified mitochondrial DNA polymerase are summarized in Table I. Omission of DNA, enzyme, or MgCl: from the incubation mixture resulted in a complete loss of activity. When three deoxy-
TABLE I REQUIREMENTS OF MOUSE LIVER MITOCHONDRIAL DNA POLYMERASE The
assay
conditions
were
as d e s c r i b e d
in Materials a n d M e t h o d s
Reactions
Complete reaction --DNA - - M g 2+ --Enzyme --dCTP, dGTP, dATP --mercaptoethanol --mezcaptoethanol + 0.03 mM N-ethylmaleimide - - m e r c a p t o e t h a n o l + 0.3 m M N - e t h y l m a l e i m i d e --activated DNA + native DNA (160 nmol) - - a c t i v a t e d D N A + single s t r a n d e d D N A ( 1 6 0 n m o l ) +DNAase * +RNAase *
except
for the modifications
listed.
[ 3 H ] TMP i n c o r p o r a t e d pmol/h
P e r c e n t a g e of complete reaction
221 0 2 0 97 159 133 69 11 53 3 224
100 0 1 0 44 72 60 31 5 24 1 100
* T h e perchloric acid insoluble product of these reactions was washed and suspended in 1 m l 0.01 M Tris, p H 7.4, containing 0.01 M M g C l 2 and incubated with either 1 0 0 ~g R N A a s e (previously boiled for 10 min) or 1 0 0 ~g D N A a s e for 60 m i n at 37°C. T h e incubation was terminated by the addition of 0.5 ml cold I M perchloric acid containing 0.02 M s o d i u m pyrophosphate and the precipitates were w a s h e d as described for tb~ D N A polymerase assay.
65 ribonucleoside triphosphates were removed from the reaction mixture, up to 44% of the control activity remained. A similar observation has been reported by Kalf et al. [19] for a rat liver mitochondrial DNA polymerase and for a mitochondrial-like DNA polymerase from ejaculated bovine spermatozoa [ 12]. The mitochondrial DNA polymerase is not dependent upon fi-mercaptoethanol for its activity although omission of the fi-mercaptoethanol resulted in a decrease of enzyme activity to 72% of the control value. Furthermore, concentrations of 0.03 and 0.3 mM N-ethylmaleimide reduced activity to 60 and 31% of control. Similar observations have been made for the rat liver and HeLa mitochondrial DNA polymerases [6,9]. At this level of purification, activated DNA served as the best natural template. The mitochondrial DNA polymerase has a limited ability to utilize single stranded template and does not significantly copy native DNA. Heat denatured single stranded template gave 24% of the activity obtained with activated DNA while native DNA template gave 2%. Meyer and Simpson [6] have reported the rat liver mitochondrial DNA polymerase shows a preference for single stranded DNA over native DNA while HeLa mitochondrial DNA polymerase best utilizes activated DNA [9]. The pH optimum of the murine mitochondrial DNA polymerase is in the range of 7.8--8.8 when assayed with activated calf thymus DNA and Tris. HC1 buffer.
Properties o f the murine mitochondrial DNA polymetase The partially purified murine mitochondrial DNA polymerase has the following properties: (1) Salt stimulation. One unique property that distinguishes the mitochondrial DNA polymerase obtained from rat liver from the HeLa cell mitochondrial enzyme is the striking stimulation of the former by salt [4,6]. A similar six- to seven-fold stimulation by 0.15 M KC1 or NaC1 was also observed with the murine mitochondrial DNA polymerase (Table II). Although the enzyme used to study the effect of salt (Table II) was partially purified by DEAE-cellulose and phosphocellulose chromatography, a similar salt stimulation response
T A B L E II T H E E F F E C T O F KC1 O N T H E M O U S E L I V E R M I T O C H O N D R I A L D N A P O L Y M E R A S E T h e r e a c t i o n m i x t u r e a n d c o n d i t i o n s o f i n c u b a t i o n w e r e as d e s c r i b e d e x c e p t t h a t t h e final KC1 c o n c e n t r a t i o n was varied. 1 0 0 % i n c o r p o r a t i o n was 2 0 7 p m o l i n c o r p o r a t e d f n .
KC1 c o n c e n t r a t i o n
[ 3H] T M P i n c o r p o r a t e d ( e x p r e s s e d as % of OM KCI)
0 0.033 0.066 0.10 0.133 0.15 0.20 0.25 0.30 0.35
100 280 513 607 687 602 485 176 73 9
66 is observed with crude extracts or after further purification of the mitochondrial DNA polymerase by gel filtration or sucrose gradient centrifugation. This degree of stimulation by salt reported here is not seen with any other murine DNA polymerase although the low molecular weight DNA polymerase (/3) is stimulated up to two-fold by KC1 concentrations up to 300 mM KC1 [20,15]. This p h e n o m e n o n is not likely to be due solely to the effect of ionic strength since different salts have been reported to show different optimal concentrations and magnitudes of effect [6]. (2) Effects of inhibitors. The mitochondrial DNA polymerase reaction has been shown to be considerably more sensitive to inhibition by ethidium bromide than most other cellular DNA polymerase [5,8]. Although care must be taken in interpreting inhibitory effects by ethidium bromide [21], in contrast to the nuclear and cytosol DNA polymerases from murine cells the activity of the mitochondrial DNA polymerase was greatly reduced by the presence of ethid.ium bromide under the conditions tested {Table III). Using 160 nmol of activated calf t h y m u s DNA as template, concentrations of 4 pM, 8 pM and 16 pM ethidium bromide reduced the activity to 43%, 29% and 19% of that of the control {Table III). Similar inhibitory effects were observed when enzyme concentrations were varied over a five-fold range. In contrast, the activity of the low and high molecular weight DNA polymerases (/3 and ~) from mouse testis or liver cells were inhibited less than 20% by concentrations of ethidium bromide up to 32 pM when assayed under identical conditions [15]. This sensitivit y to ethidium bromide for a mitochondrial DNA polymerase is in agreement with the results obtained with a mitochondrial enzyme from rat liver and h e p a t o m a [8,11]. The alkaloid antibiotic, camptothecin, has recently been shown to be a useful tool for distinguishing between several rat liver DNA polymerases [ 8 ]. In agreement with the observations of Hunter et al. [8] for a rat liver mitochondrial enzyme, camptothecin does not affect the activity of murine mitochondrial DNA polymerase at concentrations up to 100 pg/ml. (3) Inhibition by ATP. ATP can have a stimulatory or inhibitory effect on DNA synthesis [9,22]. Fry and Weissbach [9] have demonstrated that ATP is an effective inhibitor of HeLa cell mitochondrial DNA polymerase. Similar results are found with the murine mitochondrial DNA polymerase. At concenTABLE
III
INHIBITION
BY ETHIDIUM
BROMIDE
OF MURINE
MITOCHONDRIAL
DNA POLYMERASE
F r e s h l y p r e p a r e d e t h i d i u m b r o m i d e ( 2 . 5 - 1 0 - 4 M s t o c k ) w a s a d d e d at t h e c o n c e n t r a t i o n s s h o w n t o t h e s t a n d a r d r e a c t i o n m i x t u r e . In t h e e x p e r i m e n t a b o v e , 1 0 0 % i n c o r P o r a t i o n w a s 2 2 1 p m o l / h / 2 0 P l e n z y m e . S i m i l a r l e v e l s o f i n h i b i t i o n w e r e o b t a i n e d in assays in w h i c h t h e a m o u n t o f e n z y m e f r a c t i o n w a s varied u p to f i v e - f o l d . Final concentration of ethidium bromide (pM)
% Activity of control
0 4 8 16 32
100 43 29 19 6
67 trations of 2, 4 and 6 mM ATP, the enzyme activity is reduced to 60, 32 and 16%, respectively , of the control assay. This inhibition of partially purified DNA polymerases is not specific to the mitochondrial enzymes since an inhibitory effect by ATP is also seen with the cytoplasmic and nuclear DNA polymerases from HeLa cells or mouse testis [9,23]. (4) Heat inactivation. The temperature stability of the mitochondrial DNA polymerase was studied at 46°C in the presence of 1 mg/ml bovine serum albumin. The partially purified enzyme was incubated at 46°C in the absence of added template for various amounts of time after which it was cooled to 4 ° C and assayed as described in Materials and Methods. The kinetics of inactivation were nearly linear with a loss of 70%, 46% and 18% of activity after incubation times of 10, 20 and 30 min, respectively. (5) Molecular weight. The molecular weight of mitochondrial DNA polymerases is one criterion that clearly differentiates the yeast and rat liver enzymes from the HeLa cell DNA polymerase. Wintersberger and Wintersberger [2] and Probst and Meyer [11] have reported a molecular weight of 150 000 for the yeast and rat liver enzymes while Fry and Weissbach [9] find the HeLa cell enzyme to be smaller, having a molecular weight of 106 000. The molecular weight of the murine mitochondrial DNA polymerase was determined by gel filtration on Sephadex G-200 (Fig. 2). The ratio of the elution volume to the void volume, Ve : Vo, varied between 1.24 and 1.33 in six determinations. Under identical conditions lactic dehydrogenase had a Ve : Vo o f l . 3 3 - - 1 . 3 9 . Therefore, assuming a globular structure, the molecular weight of the mitochondrial DNA polymerase was estimated to between 150 000 and 170 000. Similar results were obtained when the 0.25 M KC1 was omitted from the equilibration buffer except at the lower ionic strength 10--20% of the total mitochondrial DNA polymerase was eluted in the void volume suggesting a tendency for the enzyme to aggregate. Further evidence suggesting aggregation is also seen in Fig. 3A. (6) Sedimentation coefficient. When extracted or maintained at low ionic strength, many eucaryotic DNA polymerases tend to exist in aggregate forms [15,24,25]. Previous studies analyzing crude extracts of mitochondrial DNA polymerase from mouse testis on low ionic strength sucrose gradients have revealed that the mitochondrial enzyme sedimented as a peak with a sedimentation coefficient of about 9.1 and a shoulder with an S value of approximately 10.5 [13]. The addition of 0.125 M (NH4)2SO4 to the crude extract before centrifugation did not alter the sedimentation profile. Similar results are obtained when a partially purified mitochondrial extract from liver was analyzed on a low ionic strength gradient (Fig. 3A). However, when the ionic strength of the sucrose gradient was raised to 0.25 M or 0.5 M KC1 one peak of sedimentation coefficient 8.8 S was observed. Incubation of the mitochondrial DNA polymerase with ionic detergents such as d e o x y c h o l a t e or in salt concentrations up to 8 M before sucrose gradient centrifugation failed to reduce the sedimentation coefficient below 8.8 S. Discussion
Mouse liver mitochondria contain a DNA-directed DNA polymerase that is distinguishable from the other DNA polymerases of mouse cells by differences
68 in size and enzymatic properties. The mitochondrial DNA polymerase described in this communication has a molecular weight of at least 150 000, a sedimentation coefficient of 8.8 S in the presence of up to 0.5 M KC1 and is extremely sensitive to inhibition by ethidium bromide. The activity of this enzyme is stimulated 5--6 fold by concentrations of 0.15 M KC1 or NaC1, is inhibited b y the presence of ATP, and does not require a sulfhydryl reducing agent such as fl-mercaptoethanol for activity. Furthermore, it binds to DEAEcellulose at pH 8.1, utilizes activated DNA as its most efficient natural template and cannot utilize the synthetic template, (dT) j 2 " poly(A), under the assay conditions described by Fry and Weissbach [26]. In addition to this, DNA-directed DNA polymerase, mouse cells contain several other DNA polymerases (13--15, 27--30). Using aqueous subcellular fractionation methods, mouse nuclei contain a DNA polymerase of molecular weight a b o u t 50 000 and sedimentation coefficient 3.5 S. This low molecular weight DNA polymerase is stimulated up to two-fold by the presence of KC1 and is not markedly inhibited by concentrations of ethidium bromide up to 32 pM [15]. The cytosol of aqueously subfractionated mouse cells contains two DNA polymerase activities. One, having a heterogeneous sedimentation coefficient of 6--8 S, is dissociable by salt to an activity with sedimentation coefficient 3.5 S. This enzyme has many enzymatic properties similar to the DNA polymerase fl suggesting it may be a polymeric form offi monomers [15]. The other DNA polymerase appears to be identical to the DNA polymerase a, an enzyme of sedimentation coefficient 7.2 S, which is inhibited by KC1 concentrations above 50 mM, requires fl-mercaptoethanol for activity, and is not sensitive to inhibition by ethidium bromide [ 1 5 ] . An additional class of DNA polymerase, designated R-DNA polymerases, has also been recently reported in mouse cells [ 26]. The mitochondrial DNA polymerase from mouse liver is remarkably similar to that reported for the mitochondrial enzyme from rat liver [4,6]. Meyer and Simpson [4,6,11] have reported the rat liver mitochondrial enzyme to have the following properties: a molecular weight of 150 000, a pH o p t i m u m b e t w e e n 8.0 and 9.0 with Tris buffer, adsorption to DEAE-cellulose at pH 8.0, no absolute requirement for fi-mercaptoethanol, stimulation of enzyme activity up to ten-fold by 0.15 M KC1 or NaC1 and strong inhibition of enzyme activity by ethidium bromide. Although Meyer and Simpson [6] report that rat liver enzyme favors single stranded DNA template, we find the murine enzyme preferentially utilizes activated DNA template. This difference in template utilization is not likely to be significant since they did not test activated D N A as template and template specificity varies greatly with level of purity and assay conditions for DNA polymerases. When compared to other DNA polymerases the marked stimulation of enzyme activity by ionic strength around 0.15 M could indicate a unique property of mitochondrlal DNA polymerases from rat and mouse liver [6]. However, we recently characterized the multiple forms of calf liver DNA polymerase and found that the activity of the mitochondrial enzyme from calf liver was increased up to 10-fold when assayed in the presence of 0.15 M KC1. This observation precludes the possibil.ity that the salt stimulatory effect is specific to rodent mitochondrial DNA polymerases. Furthermore, the enzyme is not
69 specific to mitochondria isolated only from liver since mitochondria from murine testis contain an enzyme with similar properties to that found in the liver [13,23], and ejaculated spermatozoa contain a DNA polymerase of 150 000 molecular weight that is also markedly stimulated by KC1 [ 1 2 ] . In contrast to the mouse, rat or calf mitochondrial DNA polymerases, the DNA polymerase from HeLa cells, an enzyme of molecular weight 106 000, does not exhibit a similar stimulation b y KC1 [9,10]. Since the reduced molecular weight and the inability for enhancement o f activity by higher ionic strength may be correlated, one possible explanation for this difference could be the loss of a c o m p o n e n t of the 150 000 molecular weight enzyme during extraction and purification. I do not, however, favor this explanation for the following reasons: (1) Several different methods of enzyme solubilization, i.e. extraction in 1 M KC1, sonication in 1 M KC1, incubation with DNAase in a low salt extraction buffer or sonication in a low salt extraction buffer yield murine mitochondrial DNA polymerases that appear similar in size, chromatographic properties and with identical responses to 0.15 M KC1. (2) The salt stimulation is observed at many levels of purification: crude extract, after DEAE-cellulose chromatography and after the DEAE-cellulose fraction is purified by phosphocellulose chromatography, gel filtration or sucrose gradient centrifugation. (3) Attempts to alter either the size or salt stimulation properties of the mouse liver enzyme by treatments with ionic detergents or salt up to 8 M have been unsuccessful and (4) The yeast mitochondrial DNA polymerase, an enzyme of molecular weight 150 000, is not stimulated by KC1. An alternative explanation for the difference in properties between the mitochondrial enzymes would support the existence of more than one DNA polymerase in mitochondria. Further studies of mitochondrial DNA polymerases using varied isolation and purification procedures in conjunction with subunit studies of homogeneously purified enzymes may help to explain the apparent differences between mitochondrial DNA polymerases from human cells and yeast and those from rodent or bovine sources. Acknowledgements This work was supported by N.S.F. Grant No. GB-44237. It is a pleasure to acknowledge the dedicated and expert technical assistance of Ms D o n n a Davidson. References 1 2 3 4 5 6 7 8 9 10 11 12
Wintersberger, E. (1966), Bioehem. Biophys. Res. Commun. 25, 1--8 Wintersberger, U. and Wintersberger, E. (1970), Eur. J. Biochem. 13, 20--27 Iwashima, A. and Rabinowitz, M. (1969) Biochim. Biophys. Acta 1 7 8 , 2 6 4 - - 2 7 3 Meyer, R.R. and Simpson, M.V. (1968) Proc. Natl. Acad. Sci. U.S. 6 1 , 1 3 0 - - 1 3 7 Meyer, R.R. and Simpson, M.V. (1969) Biochem. Biophys. Res. C ommun. 34, 238--244 Meyer, R.R. and Simpson, M.V. (1970) J. Biol. Chem. 245, 3 4 2 6 - - 3 4 3 5 Kalf, G.F. and Ch 'lh , J.J. (1968) J. Biol. Chem. 243, 4 9 0 4 - - 4 9 1 6 Hunter, G.R.0 Kalf, G.F. and Morris, H.P. (1973) Cancer Res. 33, 987--992 Fry, M. and Weissbach, A. (1973a) Biochemistry 12, 3 6 0 2 - - 3 6 0 8 Tibbetts, C.J.B. and Vinograd, J. (1973) J. Biol. Chem. 248, 3367--3379 ProbsL G.S. and Meyer, R.R. (1973) Biochem. Biophys. Res. C ommun. 50, 111--117 Hecht, N.B. (1974) J. Reprod. Fert. 41, 345--354
70 13 H e e h t , N.B. ( 1 9 7 3 ) Biochim. Biophys. A c t a 3 1 2 , 4 7 1 - - 4 8 3 14 H e c h t , N.B. ( 1 9 7 5 a ) I s o z y m e s i. Molecular S t r u c t u r e ( M a r k e r t , C., ed.), pp. 3 9 - - 5 3 , A c a d e m i c Press, New Y o r k 15 H e c h t , N.B. ( 1 9 7 5 b ) Biochim. Biophys. Acta, 3 8 3 , 3 8 8 - - 3 9 8 16 Burgess, R . R . ( 1 9 6 9 ) J. Biol. C h e m . 2 4 4 , 6 1 6 0 - - 6 1 7 0 17 Martin, R.G. a n d Ames, B.N. ( 1 9 6 1 ) J. Biol. C h e m . 2 3 6 , 1 3 7 2 - - 1 3 7 9 18 A p o s h i a n , H.V. a n d K o r n b e r g , A. ( 1 9 6 2 ) J. Biol. Chem. 2 3 7 , 5 1 9 - - 5 2 5 19 Kalf, G.F., D ' A g o s t i n o , M.A. a n d H u n t e r , G.R. ( 1 9 7 1 ) C a n c e r Res. 31, 2 0 5 4 - - 2 0 5 8 20 C h a n g , L. ( 1 9 7 3 ) J. Biol. C h e m . 2 4 8 , 3 7 8 9 - - 3 7 9 5 21 Crerar, M. and P e a r l m a n , R.E. ( 1 9 7 4 ) J. Biol. Chem. 2 4 9 , 3 1 2 3 - - 3 1 3 1 22 Wickner, W. and K o r n b e r g , A. ( 1 9 7 4 ) J. Biol. Chem. 2 4 9 , 6 2 4 4 - - 6 2 5 4 23 H e c h t , N.B., u n p u b l i s h e d o b s e r v a t i o n s 2 4 S m i t h , R.G. a n d Gallo, R.C. ( 1 9 7 2 ) Proc. Natl. A c a d . Sci., U.S. 69, 2 8 7 9 - - 2 8 8 4 2 5 Lazarus, L.H. a n d K i t r o n , N. ( 1 9 7 3 ) J. Mol. Biol. 8 1 , 5 2 9 - - 5 3 4 26 F r y , M. and Weissbach, A. ( 1 9 7 3 b ) J. Biol. Chem. 2 4 8 , 2 6 7 8 - - 2 6 8 8 27 Chang, L.M.S., B r o w n , M. a n d Bollum, F.J. ( 1 9 7 3 ) J. Mol. Biol. 74, 1--8 28 Persico, F.J., Nieholson, D.E. a n d G o t t l i e b , A.A. ( 1 9 7 3 ) C a n c e r Res. 33, 1 2 1 0 - - 1 2 1 6 29 A d a m s , R.L.P., H e n d e r s o n , M.A.L., W o o d , W. a n d L i n d s a y , J. ( 1 9 7 3 ) B i o c h e m . J. 1 3 1 , 2 3 7 - - 2 4 6 30 Matsukage, A.. B o h n , E.W. a n d Wilson, S.H. ( 1 9 7 4 ) Proc. Natl. Acad. Sci. U.S. 7 1 , 5 7 8 - - 5 8 5
Biochimica et Biophysica Acta, 414 (1975) 59--70 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 98447
A DNA-DIRECTED DNA POLYMERASE FROM MURINE L I V E R MITOCHONDRIA
NORMAN B. HECHT
Department of Biology, Tufts University, Medford, Mass. 02155 (U.S.A.) (Received June 3rd, 1975)
Summary A DNA-directed DNA polymerase has been isolated from murine liver mitochondria. The mitochondrial DNA polymerase is distinguishable from other DNA polymerases found in the nucleus and cytosol of murine cells by several enzymatic and physical properties. It is stimulated 5--6-fold by 0.15 M KC1, does not require a sulfhydryl reducing agent for activity, and is inhibited by ethidium bromide or ATP. The enzyme has a sedimentation coefficient of 8.8 S in the presence of up to 0.5 M KC1, a molecular weight of 150--170 000, and utilizes natural templates in the following order of preference: activated DNA (100%), single stranded DNA (24%), and native DNA (5%).
Introduction In addition to containing a unique DNA, mitochondria isolated from several different sources have been shown to possess a unique DNA polymerase. This enzyme has been reported to differ from the DNA polymerases found in the nuclear and cytosol fractions of the cell by the criteria of: molecular weight, sensitivity to inhibitors, chromatographic behavior, primer-template utilization, and requirements for maximal activity [ 1--10]. Despite the general agreement for the existence of a distinct DNA polymerase in mitochondria, a comparison of some of the properties o f the enzymes isolated from yeast, rat liver, and HeLa cells suggests differences between mitochondrial DNA polymerases. For example, the molecular weight has been reported to be a b o u t 150 000 for the rat liver and yeast mitochondrial D N A polymerases while the HeLa cell enzyme has a molecular weight of 106 000 [2,9,11]. In addition, the enzymatic activity o f crude or highly purified rat liver mitochondrial DNA polymerase is dramatically stimulated by ionic strengths of 0.15 M KC1 whereas the mitochondrial enzymes isolated from HeLa cells or yeast do not show a similar response to elevated ionic strength [2,4,6,9,10]. These observations and the
60 fact that we wished to relate a DNA-directed DNA polymerase isolated from mature spermatozoa [12] to other cellular D N A polymerases p r o m p t e d us to examine the DNA polymerase activity found in purified mitochondria. After aqueous subcellular fractionation, murine liver cells contain a low molecular weight DNA-directed DNA polymerase (~) in the nuclear fraction and two distinguishable DNA-directed DNA polymerases in the cytosol [13-15]. The cytosol enzymes are distinguishable by the criteria of salt sensitivity, chromatographic properties, inhibition by N-ethylmaleimide, template preference, and size [ 1 3 - - 1 5 ] . One cytosol enzyme, dissociable by 0.25 M KC1 to an active form of DNA polymerase of sedimentation coefficient 3.5 S, shares many chemical/physical properties with the DNA polymerase ~ suggesting it may represent a polymeric form of ~ monomers. The size, response to physiological change, and reaction properties of the other cytosol DNA polymerase suggest it is the DNA polymerase ~ [ 1 5 ] . In this communication I describe the properties of a DNA-directed DNA polymerase isolated from purified mitochondria of murine liver. This enzyme differs from other murine DNA polymerases found in the nucleus or cytosol by several criteria. When compared to the mitochondrial DNA polymerases from yeast, HeLa cells, and rat liver, it most closely resembles the enzyme from rat liver. Materials and Methods
Materials [ 3H] TTP (spec. act. 40 Ci/mmol) was purchased from New England Nuclear, Boston, Mass. Schwarz-Mann, Orangeburg, N.Y. was the supplier of dATP, dGTP, dTTP, dCTP and enzyme grade sucrose. Ethidium bromide, calf thymus DNA, and fraction IV bovine serum albumin were purchased from Sigma Chemical Co., St. Louis, Missouri. Phosphocellulose (P-11) was a p r o d u c t of Whatman, Co., U.K. Sephadex G-200 was obtained from Pharmacia, Inc., Piscataway, N.J. Diethylaminoethyl cellulose (Cellex-D) was purchased from Bio Rad Laboratories, Richmond, Cal. The sodium salt of camptothecin was generously provided by Dr Harry B. Woods of the Cancer Chemotherapy Branch of N.I.H. Mice (mature male CD-1 strain) were obtained from Charles River Breeding Laboratories, Inc., Wilmington, Mass. Preparation of mi tochondria Mice were killed by cervical dislocation, the livers were removed and immediately washed in suspension buffer {0.25 M sucrose containing 0.02 M Tris, pH 8.1, 1 mM CaC12, 3 mM ~-mercaptoethanol) at 4°C. The livers were minced, washed several times more with the suspension buffer, homogenized b y hand in a Teflon and glass homogenizer (10 X), and filtered through 4 layers of cheese cloth. The nuclear fraction was removed by centrifugation of the filtrate at 650 X g for 15 min. in a Sorvall HB-4 swinging bucket rotor and the supernatant was centrifuged at 13 000 X g for 30 min. The pellet, containing mitochondria, was resuspended in suspension buffer and recentrifuged at 13 000 X g for 30 min. This recentrifugation step was repeated three times. The final pellet was resuspended and layered over a linear sucrose gradient of
61 30% to 65% sucrose (dissolved in suspension buffer) and centrifuged for 90 min. at 24 000 rev./min in a SW27 rotor in a Beckman L2-65 ultracentrifuge. The mitochondrial band was removed, washed with suspension buffer, pelleted, and either used immediately or stored in liquid nitrogen. In a typical preparation 1 g of mouse liver (wet weight) yielded approximately 2 mg of mitochondrial protein.
Preparation o f mitochondrial DNA polymerase extract Frozen or fresh mitochondria were suspended in extraction buffer (0.02 M Tris, pH 8.1, 0.05 M NaC1, 3 mM fi-mercaptoethanol, 1 mM EDTA, 20% ethylene glycol) containing 1 M KC1 and sonicated for 60 s at a setting of 4 with a Branson sonicator (Model 185E) followed by incubation at 4°C for 60 min. The extract was centrifuged at 105 000 X g for 60 min in a'Beckman type 65 rotor and the supernatant was decanted and dialyzed overnight against extraction buffer. This salt extraction procedure was effective in solubilizing over 96% of the DNA polymerase activity. DEAE-cellulose chromatography Cellex-D was prepared as previously described [13] and equilibrated with extraction buffer. The dialyzed enzyme extract containing 18 mg of protein was adsorbed onto a DEAE-cellulose column at a proportion of 1 mg of protein/g of Cellex-D. The column was washed with several column volumes of the same buffer and eluted with a linear gradient from 0.05 M to 0.55 M NaC1 containing 0.02 M Tris, pH 8.1, 3 mM fi-mercaptoethanol, 1 mM EDTA, and 20% ethylene glycol. Fractions (0.7 ml) were collected directly into bovine serum' albumin (1 mg/ml) and aliquots (0.02 ml) were assayed for DNA polymerase activity. Phosphocellulose chromatography o f the mitochondrial extract Phosphocellulose was washed by the method of Burgess [ 16]. The pooled peak fractions of the DNA polymerase obtained from the DEAE-cellulose column (see Results) were dialyzed against extraction buffer and adsorbed onto phosphocellulose columns (1 X 15 cm) that had been equilibrated with extraction buffer. The columns were washed with several bed volumes of this buffer and then eluted with linear gradients from 0 to 1.0 M KC! in extraction buffer. Fractions (0.75 ml) were collected directly into bovine serum albumin (1 mg/ml final concentration) and aliquots (0.02 ml) were assayed for DNA polymerase activity. Gel filtration o f mitochondrial DNA polymerase An aliquot (1 ml) of th~ pooled peak tubes obtained from phosphocellulose chromatography (tubes 15--18 of Fig. 1) was dialyzed and layered on a Sephadex C~200 column (0.8 X 50 cm) equilibrated with extraction buffer containing 0.25 M KC1. The exclusion volume (V0) was determined by blue dextran marker and the column was calibrated with catalase, lactic dehydrogenase, alkaline phosphatase, ovalbumin, and cytochrome c.
62 5o! J 40
'o
~0
o
_~ 2o rzl
5
I0 15 FRACTION NUMBER
20
25
Fig. 1. P h o s p h o e e n u l o s e c h r o m a t o g r a p h y o f an e x t r a c t o f m u r i n e l i v e r m i t o c h o n d r i a . T h e f r a c t i o n s c o n r a i n i n g t h e D N A p o l y m e r a s e p e a k o b t a i n e d b y D E A E - c e l l u l o s e c h r o m a t o g r a p h y a n d c o n t a i n i n g 14 m g of p r o t e i n w e r e p o o l e d , d i a l y z e d a n d c h r o m a t o g r a p h e d o n a 1 × 15 c m p h o s p h o c e l l u l o s e c o l u m n . E l u t i o n w a s c a r r i e d o u t as d e s c r i b e d u n d e r M a t e r i a l s a n d M e t h o d s a n d a l i q u o t s ( 0 . 0 2 m l ) w e r e a s s a y e d f o r D N A p o l y m e r a s e as described.
Sucrose gradient cen trifuga tion Sedimentation coefficients were determined using the m e t h o d of Martin and Ames [17]. A dialyzed aliquot (0.2 ml} from the peak tubes of the phosphocellulose column was layered over preformed 5 to 20% sucrose gradients (sucrose dissolved in extraction buffer or extraction buffer + 0.5 M KC1) and centrifuged for 16 h at 55 000 rev./min at 4°C in a SW 65 rotor in a Beckman L2-65. Aliquots (0.01 ml) of alkaline phosphatase (S20,w = 6.3) and catalase ($20,w = 11.3) were added to the DNA polymerase and run as marker proteins. Assay conditions for mitochondrial D N A polymerase The standard reaction mixture (0.3 ml) contained 5 pmol Tris • HC1 buffer at pH 8.8, 2.5 pmol MgC12, 3 pmol fl-mercaptoethanol, 40 pmol KC1, 160 nmol activated calf t h y m u s DNA, 10 nmol each of dCTP, dGTP, dATP, 1 nmol of [ZH]TTP (100--750 cpm/pmol) and enzyme. Incubation was for 60 min at 37 °C. The calf t h y m u s DNA was activated by the m e t h o d of Aposhian and Kornberg [18]. After incubation, the assay tubes were placed in ice and the reaction was stopped by the addition of 0.5 ml cold 1 M HC104 containing 0.02 M sodium pyrophosphate. The precipitates were collected on glass fiber filters (Whatman GF/A) and washed with 10 ml of 5% HC104 containing 0.02 M sodium pyrophosphate and with 10 ml of 95% ethanol. The filters were dried and the bound radioactivity was counted in a liquid scintillation counter. All assays were performed in duplicate.
63 Results
Partial purification of DNA polymerase from murine liver mitochondria The procedure for extracting the murine mitochondria described under Materials and Methods solubilized over 96% of the total DNA-directed DNA polymerase but only released 20--25% of the total mitochondrial protein. The combination of high ionic strength (1 M KC1) and sonication seems essential for maximal release of the DNA polymerase since sonication at lower ionic strengths (extraction buffer in the absence of 1 M KC1) gave variable results, often releasing less than 50% of the total DNA polymerase activity from the mitochondrial pellet. Extraction of the mitochondria at 1 M KC1 in the absence o f sonication allowed 20--40% of the enzyme to remain in the pellet fraction. After dialysis, the mitochondrial extract was chromatographed on a DEAE-cellulose column. Under the conditions used, the DNA polymerase b o u n d quantitatively and upon application of a salt gradient eluted at 0.24 M NaC1. These results are identical to those we previously reported for a mitochondrial DNA polymerase from mouse testis [13]. Recoveries of enzyme activity ranged between 88 and 106% o f the activity from the initial mitochondrial extract. It was essential to collect the DNA polymerase eluted from either DEAE-cellulose or phosphocellulose columns directly into bovine serum albu20
1
A
%
IO
,6
8
5
s
o_
~
2o
z_
Z Io
5
I0 15 VOLUME (ML)
20
25
5
IO 15 FRACTION NUMBER
20
25
Fig. 2. Gel f i l t r a t i o n of d i a l y z e d f r a c t i o n s f r o m a m i t o c h o n d r i a l liver p h o s p h o c e l l u l o s c c o l u m n . T h e fractions containing the peak of DNA polymerase activity from phosphocelhdose columns were pooled, d i a l y z e d , a n d 1.0 m l was l a y e r e d o v e r a 0.8 X 50 c m S e p h a d e x G - 2 0 0 c o l u m n t h a t w a s e q u i l i b r a t e d w i t h e x t r a c t i o n b u f f e r c o n t a i n i n g 0 . 2 5 M KCI. F r a c t i o n ( 0 . 5 m l ) w e r e c o l l e c t e d a n d 0 . 0 5 m l s a m p l e s w e r e a s s a y e d f o r D N A p o l y m e r a s e . T h e c o l u m n w a s c a l i b r a t e d as d e s c r i b e d in Materials and M e t h o d s . R o u t i n e ly 4 0 - - 6 0 % o f t h e l o a d e d D N A p o l y m e r a s c was r e c o v e r e d . Fig. 3. S u c r o s e d e n s i t y g r a d i e n t cellulose c o l u m n . A l i q u o t s (0.2 layered over p r e f o r m e d sucrose t i o n b u f f e r ; B, s u c r o s e dissolved
c e n t r i f u g a t i o n o f d i a l y z e d f r a c t i o n s f r o m a m i t o c h o n d r i a l liver p h o s p h o ml) from the peak tubes o f a phosphocellulose c o l u m n were dialyzed and g r a d i e n t s . S e d i m e n t a t i o n w a s to t h e right. A, s u c r o s e dissolved in e x t r a c in e x t r a c t i o n b u f f e r c o n t a i n i n g 0.5 M KC1.
64 min to maintain good recoveries (greater than 85%) o f the loaded DNA polymerase. If bovine serum albumin was omitted, a maximum recovery of 50% was obtained. This has made it difficult to accurately calculate specific activities during the partial purification stages b u t has ensured that the enzyme fraction that was eluted from phosphocellulose represents between 80 and 90% o f the total activity from the initial mitochondrial extract. The reason that bovine serum albumin serves to increase the recovery of the mitochondrial D N A polymerase is not clear. However, it may help to reduce the "stickiness" o f the DNA polymerase since this enzyme tends to stick to glass quite readily and also aggregates in low ionic strength extraction buffers (Fig. 3A). The peak tubes elu-ting at 0.24 M NaC1 from the DEAE-cellulose were combined, dialyzed and further purified by phosphocellulose chromatography (Fig. 1). The DNA polymerase activity bound quantitatively and eluted as a sharp peak at 0.56 M KC1. The recovery of the DNA polymerase from the phosphocellulose column was nearly quantitative representing a recovery of 81--92% of the DNA polymerase from the mitochondrial extract. The fractions containing enzyme were combined, dialyzed, and stored under liquid nitrogen. All further investigations concerning the requirements and properties of the murine mitochondrial DNA polymerase were carried o u t using pooled peak fractions derived from phosphocellulose columns.
Requirements of mitochondrial DNA polymerase The requirements of the partially purified mitochondrial DNA polymerase are summarized in Table I. Omission of DNA, enzyme, or MgCl: from the incubation mixture resulted in a complete loss of activity. When three deoxy-
TABLE I REQUIREMENTS OF MOUSE LIVER MITOCHONDRIAL DNA POLYMERASE The
assay
conditions
were
as d e s c r i b e d
in Materials a n d M e t h o d s
Reactions
Complete reaction --DNA - - M g 2+ --Enzyme --dCTP, dGTP, dATP --mercaptoethanol --mezcaptoethanol + 0.03 mM N-ethylmaleimide - - m e r c a p t o e t h a n o l + 0.3 m M N - e t h y l m a l e i m i d e --activated DNA + native DNA (160 nmol) - - a c t i v a t e d D N A + single s t r a n d e d D N A ( 1 6 0 n m o l ) +DNAase * +RNAase *
except
for the modifications
listed.
[ 3 H ] TMP i n c o r p o r a t e d pmol/h
P e r c e n t a g e of complete reaction
221 0 2 0 97 159 133 69 11 53 3 224
100 0 1 0 44 72 60 31 5 24 1 100
* T h e perchloric acid insoluble product of these reactions was washed and suspended in 1 m l 0.01 M Tris, p H 7.4, containing 0.01 M M g C l 2 and incubated with either 1 0 0 ~g R N A a s e (previously boiled for 10 min) or 1 0 0 ~g D N A a s e for 60 m i n at 37°C. T h e incubation was terminated by the addition of 0.5 ml cold I M perchloric acid containing 0.02 M s o d i u m pyrophosphate and the precipitates were w a s h e d as described for tb~ D N A polymerase assay.
65 ribonucleoside triphosphates were removed from the reaction mixture, up to 44% of the control activity remained. A similar observation has been reported by Kalf et al. [19] for a rat liver mitochondrial DNA polymerase and for a mitochondrial-like DNA polymerase from ejaculated bovine spermatozoa [ 12]. The mitochondrial DNA polymerase is not dependent upon fi-mercaptoethanol for its activity although omission of the fi-mercaptoethanol resulted in a decrease of enzyme activity to 72% of the control value. Furthermore, concentrations of 0.03 and 0.3 mM N-ethylmaleimide reduced activity to 60 and 31% of control. Similar observations have been made for the rat liver and HeLa mitochondrial DNA polymerases [6,9]. At this level of purification, activated DNA served as the best natural template. The mitochondrial DNA polymerase has a limited ability to utilize single stranded template and does not significantly copy native DNA. Heat denatured single stranded template gave 24% of the activity obtained with activated DNA while native DNA template gave 2%. Meyer and Simpson [6] have reported the rat liver mitochondrial DNA polymerase shows a preference for single stranded DNA over native DNA while HeLa mitochondrial DNA polymerase best utilizes activated DNA [9]. The pH optimum of the murine mitochondrial DNA polymerase is in the range of 7.8--8.8 when assayed with activated calf thymus DNA and Tris. HC1 buffer.
Properties o f the murine mitochondrial DNA polymetase The partially purified murine mitochondrial DNA polymerase has the following properties: (1) Salt stimulation. One unique property that distinguishes the mitochondrial DNA polymerase obtained from rat liver from the HeLa cell mitochondrial enzyme is the striking stimulation of the former by salt [4,6]. A similar six- to seven-fold stimulation by 0.15 M KC1 or NaC1 was also observed with the murine mitochondrial DNA polymerase (Table II). Although the enzyme used to study the effect of salt (Table II) was partially purified by DEAE-cellulose and phosphocellulose chromatography, a similar salt stimulation response
T A B L E II T H E E F F E C T O F KC1 O N T H E M O U S E L I V E R M I T O C H O N D R I A L D N A P O L Y M E R A S E T h e r e a c t i o n m i x t u r e a n d c o n d i t i o n s o f i n c u b a t i o n w e r e as d e s c r i b e d e x c e p t t h a t t h e final KC1 c o n c e n t r a t i o n was varied. 1 0 0 % i n c o r p o r a t i o n was 2 0 7 p m o l i n c o r p o r a t e d f n .
KC1 c o n c e n t r a t i o n
[ 3H] T M P i n c o r p o r a t e d ( e x p r e s s e d as % of OM KCI)
0 0.033 0.066 0.10 0.133 0.15 0.20 0.25 0.30 0.35
100 280 513 607 687 602 485 176 73 9
66 is observed with crude extracts or after further purification of the mitochondrial DNA polymerase by gel filtration or sucrose gradient centrifugation. This degree of stimulation by salt reported here is not seen with any other murine DNA polymerase although the low molecular weight DNA polymerase (/3) is stimulated up to two-fold by KC1 concentrations up to 300 mM KC1 [20,15]. This p h e n o m e n o n is not likely to be due solely to the effect of ionic strength since different salts have been reported to show different optimal concentrations and magnitudes of effect [6]. (2) Effects of inhibitors. The mitochondrial DNA polymerase reaction has been shown to be considerably more sensitive to inhibition by ethidium bromide than most other cellular DNA polymerase [5,8]. Although care must be taken in interpreting inhibitory effects by ethidium bromide [21], in contrast to the nuclear and cytosol DNA polymerases from murine cells the activity of the mitochondrial DNA polymerase was greatly reduced by the presence of ethid.ium bromide under the conditions tested {Table III). Using 160 nmol of activated calf t h y m u s DNA as template, concentrations of 4 pM, 8 pM and 16 pM ethidium bromide reduced the activity to 43%, 29% and 19% of that of the control {Table III). Similar inhibitory effects were observed when enzyme concentrations were varied over a five-fold range. In contrast, the activity of the low and high molecular weight DNA polymerases (/3 and ~) from mouse testis or liver cells were inhibited less than 20% by concentrations of ethidium bromide up to 32 pM when assayed under identical conditions [15]. This sensitivit y to ethidium bromide for a mitochondrial DNA polymerase is in agreement with the results obtained with a mitochondrial enzyme from rat liver and h e p a t o m a [8,11]. The alkaloid antibiotic, camptothecin, has recently been shown to be a useful tool for distinguishing between several rat liver DNA polymerases [ 8 ]. In agreement with the observations of Hunter et al. [8] for a rat liver mitochondrial enzyme, camptothecin does not affect the activity of murine mitochondrial DNA polymerase at concentrations up to 100 pg/ml. (3) Inhibition by ATP. ATP can have a stimulatory or inhibitory effect on DNA synthesis [9,22]. Fry and Weissbach [9] have demonstrated that ATP is an effective inhibitor of HeLa cell mitochondrial DNA polymerase. Similar results are found with the murine mitochondrial DNA polymerase. At concenTABLE
III
INHIBITION
BY ETHIDIUM
BROMIDE
OF MURINE
MITOCHONDRIAL
DNA POLYMERASE
F r e s h l y p r e p a r e d e t h i d i u m b r o m i d e ( 2 . 5 - 1 0 - 4 M s t o c k ) w a s a d d e d at t h e c o n c e n t r a t i o n s s h o w n t o t h e s t a n d a r d r e a c t i o n m i x t u r e . In t h e e x p e r i m e n t a b o v e , 1 0 0 % i n c o r P o r a t i o n w a s 2 2 1 p m o l / h / 2 0 P l e n z y m e . S i m i l a r l e v e l s o f i n h i b i t i o n w e r e o b t a i n e d in assays in w h i c h t h e a m o u n t o f e n z y m e f r a c t i o n w a s varied u p to f i v e - f o l d . Final concentration of ethidium bromide (pM)
% Activity of control
0 4 8 16 32
100 43 29 19 6
67 trations of 2, 4 and 6 mM ATP, the enzyme activity is reduced to 60, 32 and 16%, respectively , of the control assay. This inhibition of partially purified DNA polymerases is not specific to the mitochondrial enzymes since an inhibitory effect by ATP is also seen with the cytoplasmic and nuclear DNA polymerases from HeLa cells or mouse testis [9,23]. (4) Heat inactivation. The temperature stability of the mitochondrial DNA polymerase was studied at 46°C in the presence of 1 mg/ml bovine serum albumin. The partially purified enzyme was incubated at 46°C in the absence of added template for various amounts of time after which it was cooled to 4 ° C and assayed as described in Materials and Methods. The kinetics of inactivation were nearly linear with a loss of 70%, 46% and 18% of activity after incubation times of 10, 20 and 30 min, respectively. (5) Molecular weight. The molecular weight of mitochondrial DNA polymerases is one criterion that clearly differentiates the yeast and rat liver enzymes from the HeLa cell DNA polymerase. Wintersberger and Wintersberger [2] and Probst and Meyer [11] have reported a molecular weight of 150 000 for the yeast and rat liver enzymes while Fry and Weissbach [9] find the HeLa cell enzyme to be smaller, having a molecular weight of 106 000. The molecular weight of the murine mitochondrial DNA polymerase was determined by gel filtration on Sephadex G-200 (Fig. 2). The ratio of the elution volume to the void volume, Ve : Vo, varied between 1.24 and 1.33 in six determinations. Under identical conditions lactic dehydrogenase had a Ve : Vo o f l . 3 3 - - 1 . 3 9 . Therefore, assuming a globular structure, the molecular weight of the mitochondrial DNA polymerase was estimated to between 150 000 and 170 000. Similar results were obtained when the 0.25 M KC1 was omitted from the equilibration buffer except at the lower ionic strength 10--20% of the total mitochondrial DNA polymerase was eluted in the void volume suggesting a tendency for the enzyme to aggregate. Further evidence suggesting aggregation is also seen in Fig. 3A. (6) Sedimentation coefficient. When extracted or maintained at low ionic strength, many eucaryotic DNA polymerases tend to exist in aggregate forms [15,24,25]. Previous studies analyzing crude extracts of mitochondrial DNA polymerase from mouse testis on low ionic strength sucrose gradients have revealed that the mitochondrial enzyme sedimented as a peak with a sedimentation coefficient of about 9.1 and a shoulder with an S value of approximately 10.5 [13]. The addition of 0.125 M (NH4)2SO4 to the crude extract before centrifugation did not alter the sedimentation profile. Similar results are obtained when a partially purified mitochondrial extract from liver was analyzed on a low ionic strength gradient (Fig. 3A). However, when the ionic strength of the sucrose gradient was raised to 0.25 M or 0.5 M KC1 one peak of sedimentation coefficient 8.8 S was observed. Incubation of the mitochondrial DNA polymerase with ionic detergents such as d e o x y c h o l a t e or in salt concentrations up to 8 M before sucrose gradient centrifugation failed to reduce the sedimentation coefficient below 8.8 S. Discussion
Mouse liver mitochondria contain a DNA-directed DNA polymerase that is distinguishable from the other DNA polymerases of mouse cells by differences
68 in size and enzymatic properties. The mitochondrial DNA polymerase described in this communication has a molecular weight of at least 150 000, a sedimentation coefficient of 8.8 S in the presence of up to 0.5 M KC1 and is extremely sensitive to inhibition by ethidium bromide. The activity of this enzyme is stimulated 5--6 fold by concentrations of 0.15 M KC1 or NaC1, is inhibited b y the presence of ATP, and does not require a sulfhydryl reducing agent such as fl-mercaptoethanol for activity. Furthermore, it binds to DEAEcellulose at pH 8.1, utilizes activated DNA as its most efficient natural template and cannot utilize the synthetic template, (dT) j 2 " poly(A), under the assay conditions described by Fry and Weissbach [26]. In addition to this, DNA-directed DNA polymerase, mouse cells contain several other DNA polymerases (13--15, 27--30). Using aqueous subcellular fractionation methods, mouse nuclei contain a DNA polymerase of molecular weight a b o u t 50 000 and sedimentation coefficient 3.5 S. This low molecular weight DNA polymerase is stimulated up to two-fold by the presence of KC1 and is not markedly inhibited by concentrations of ethidium bromide up to 32 pM [15]. The cytosol of aqueously subfractionated mouse cells contains two DNA polymerase activities. One, having a heterogeneous sedimentation coefficient of 6--8 S, is dissociable by salt to an activity with sedimentation coefficient 3.5 S. This enzyme has many enzymatic properties similar to the DNA polymerase fl suggesting it may be a polymeric form offi monomers [15]. The other DNA polymerase appears to be identical to the DNA polymerase a, an enzyme of sedimentation coefficient 7.2 S, which is inhibited by KC1 concentrations above 50 mM, requires fl-mercaptoethanol for activity, and is not sensitive to inhibition by ethidium bromide [ 1 5 ] . An additional class of DNA polymerase, designated R-DNA polymerases, has also been recently reported in mouse cells [ 26]. The mitochondrial DNA polymerase from mouse liver is remarkably similar to that reported for the mitochondrial enzyme from rat liver [4,6]. Meyer and Simpson [4,6,11] have reported the rat liver mitochondrial enzyme to have the following properties: a molecular weight of 150 000, a pH o p t i m u m b e t w e e n 8.0 and 9.0 with Tris buffer, adsorption to DEAE-cellulose at pH 8.0, no absolute requirement for fi-mercaptoethanol, stimulation of enzyme activity up to ten-fold by 0.15 M KC1 or NaC1 and strong inhibition of enzyme activity by ethidium bromide. Although Meyer and Simpson [6] report that rat liver enzyme favors single stranded DNA template, we find the murine enzyme preferentially utilizes activated DNA template. This difference in template utilization is not likely to be significant since they did not test activated D N A as template and template specificity varies greatly with level of purity and assay conditions for DNA polymerases. When compared to other DNA polymerases the marked stimulation of enzyme activity by ionic strength around 0.15 M could indicate a unique property of mitochondrlal DNA polymerases from rat and mouse liver [6]. However, we recently characterized the multiple forms of calf liver DNA polymerase and found that the activity of the mitochondrial enzyme from calf liver was increased up to 10-fold when assayed in the presence of 0.15 M KC1. This observation precludes the possibil.ity that the salt stimulatory effect is specific to rodent mitochondrial DNA polymerases. Furthermore, the enzyme is not
69 specific to mitochondria isolated only from liver since mitochondria from murine testis contain an enzyme with similar properties to that found in the liver [13,23], and ejaculated spermatozoa contain a DNA polymerase of 150 000 molecular weight that is also markedly stimulated by KC1 [ 1 2 ] . In contrast to the mouse, rat or calf mitochondrial DNA polymerases, the DNA polymerase from HeLa cells, an enzyme of molecular weight 106 000, does not exhibit a similar stimulation b y KC1 [9,10]. Since the reduced molecular weight and the inability for enhancement o f activity by higher ionic strength may be correlated, one possible explanation for this difference could be the loss of a c o m p o n e n t of the 150 000 molecular weight enzyme during extraction and purification. I do not, however, favor this explanation for the following reasons: (1) Several different methods of enzyme solubilization, i.e. extraction in 1 M KC1, sonication in 1 M KC1, incubation with DNAase in a low salt extraction buffer or sonication in a low salt extraction buffer yield murine mitochondrial DNA polymerases that appear similar in size, chromatographic properties and with identical responses to 0.15 M KC1. (2) The salt stimulation is observed at many levels of purification: crude extract, after DEAE-cellulose chromatography and after the DEAE-cellulose fraction is purified by phosphocellulose chromatography, gel filtration or sucrose gradient centrifugation. (3) Attempts to alter either the size or salt stimulation properties of the mouse liver enzyme by treatments with ionic detergents or salt up to 8 M have been unsuccessful and (4) The yeast mitochondrial DNA polymerase, an enzyme of molecular weight 150 000, is not stimulated by KC1. An alternative explanation for the difference in properties between the mitochondrial enzymes would support the existence of more than one DNA polymerase in mitochondria. Further studies of mitochondrial DNA polymerases using varied isolation and purification procedures in conjunction with subunit studies of homogeneously purified enzymes may help to explain the apparent differences between mitochondrial DNA polymerases from human cells and yeast and those from rodent or bovine sources. Acknowledgements This work was supported by N.S.F. Grant No. GB-44237. It is a pleasure to acknowledge the dedicated and expert technical assistance of Ms D o n n a Davidson. References 1 2 3 4 5 6 7 8 9 10 11 12
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