10

Biochimica et Biophysica Acta, 561 (1979) 10--16

© Elsevier/North-Holland Biomedical Press

BBA 99385

DETECTION AND C H A R A C T E R I Z A T I O N OF DNA POLYMERASE FROM TR YPANOSOMA

BR UCEI

DIPAK K. DUBE a, RICHARD O. WILLIAMS b, GITA SEAL a and SALLY C. WILLIAMS b a The Institute for Cancer Research, The Fox Chase Cancer Center, Philadelphia, PA 19111 (U.S.A.) and b International Laboratory for Research on Animal Diseases, P.O. Box 30709, Nairobi (Kenya)

(Received May 2nd, 1978) Key words: DNA polymerase; Kinetoplastida; Parasitic protozoa; (Trypanosoma brucei)

Summary The predominant DNA polymerase activity has been isolated from the parasitic flagellated protozoan, T r y p a n o s o m a brucei. Like mammalian DNA polymerase-a the trypanosome DNA polymerase is of large molecular weight (S, 6--8), is resistant to thermal denaturation, is sensitive to N-ethylmaleimide, and is inhibited by high ionic strength. However, specific antisera that crossreact with mammalian DNA polymerase-a from different species fail to crossreact with the trypanosome polymerase. Introduction

Multiple species of DNA polymerases have been described in eucaryotic cells [1--5]. In the case of mammalian cells, there is widespread agreement that three different DNA polymerases can be distinguished, in addition to mitochondrial DNA polymerase. These have been designated as DNA polymerase-~, -/3, -7, and mt (mitchondrial). However, recent evidence suggests that the DNA polymerase mt is a 7-polymerase [6]. It seems likely that this classification scheme may eventually be applicable to the DNA polymerases present in most eucaryotic cells. DNA polymerase-fi, the lower molecular weight and comparatively N-ethylmaleimide resistant species of DNA dependent DNA polymerase, has n o t been found in unicellular eucaryotes or plants [7--9]. T r y p a n o s o m a brucei, a parasitic flagellated protozoan (order kinetoplastidia), is a m e m b e r of the group of African trypanosomes known to cause sleeping sickness in humans and Nagana in cattle. We report here the isolation and characterization of DNA polymerase from T. brucei. In the present paper we show that this eucaryotic organism contains only one predominant class of polymerase activity. Although the DNA polymerase activity from T. brucei

11 resembles DNA polymerase-a from mammalian systems, it does not cross-react with the antisera against the latter. Methods

Purification of trypanosome DNA polymerase. Trypanosoma brucei (strain S 427) was grown in inbred rats and passaged by intraperitoneal infection. Blood was collected by heart puncture from rats with rising parasitemias and the trypanosomes were isolated from the blood using a modification of the method reported by Lanham and Godfrey [10]. 8 . 1 0 9 isolated trypanosomes were homogenized at 4°C with a loosely fitted Teflon homogenizer in buffer A (10 mM Tris-HC1, pH 7.4, 2.5 mM dithiothreitol/10% glycerol and 1 mM EDTA) containing 1.0 M KC1 and 0.3% Triton X-100. The homogenate was centrifuged at 20 000 × g for 30 min and the resulting supernatant solution was dialyzed against buffer B (50 mM Tris-HC1, pH 7.5, 10% glycerol) for 12 h. The specific activity of DNA polymerase in the crude supernatant solution was 0.02 units/mg protein. The dialyzed supernatant was adsorbed onto a 1 × 15 cm DEAE cellulose (DE-23) column equilibrated with 50 mM Tris-HC1, pH 7.5, 1 mM dithiothreitol and 10% glycerol and then eluted with 20 ml of the same buffer containing 0.3 M KC1. At least 90% of the nucleic acid was removed by this treatment as determined by A26onm/A2aonm ratios from aliquots of input and eluted material. Total DNA polymerase activity was increased about 30-fold after DEAE-cellulose treatment. Active fractions from DE-23 column were combined and dialyzed against buffer B for 12 h at 4°C and then were adsorbed onto a 1 × 15 cm phosphocellulose ( P l l ) column equilibrated with buffer A containing 50 mM KC1. The column was washed with 20 ml buffer A containing 70 mM KC1 and eluted with a 200 ml linear KC1 gradient (0.1-0.6 M KC1) in buffer B. Fifty fractions were collected from the column and assayed for DNA polymerase activity. A single peak of constant specific activity (3.3 units/mg protein) was eluted at 0.3 M KC1. The peak fractions were pooled and concentrated by preevaporation. The specific activity represented an increase of 165-fold over the crude extract. DNA polymerase activity. Assays were carried out in duplicate in a reaction mixture (total vol. 0.1 ml) containing: 20 mM Tris-HC1, pH 7.4/5 mM 2-mercaptoethanol/10 mM MgC12/50 mM KC1/20 gM each of dATP, dCTP, dGTP and [~_32p] or [3H]dTTP (1000--2000 cpm/pmol)/10 ~g bovine serum albumin/25 ~g activated calf thymus DNA and 0.05--0.1 units of DNA polymerase. (In the case of mammalian DNA polymerase-fl from other sources, 100 mM KC1 was present in the reaction mixture). The reactions were incubated for 15 min at 37°C and were stopped by the addition of cold perchloric acid and 100 pg calf thymus DNA. The acid insoluble precipitate was collected for determination of radioactivity as previously described [11]. One unit of DNA polymerase activity is defined as the incorporation of 1 nmol of total nucleotide per h. Materials

Radioactive deoxynucleoside triphosphates were purchased from New England Nuclear or Amersham, U.K. Phosphonoacetic acid was purchased from

12 ICN, K and K Laboratories, Cleveland, Ohio. DNA polymerase-a from HeLa cells and antibody against this enzyme was a kind gift of A. Weissnach (Roche Institute of Molecular Biology, Nutley, N.J.), antisera against homogeneous E. coli DNA polymerase I [12] and avian myloblastosis virus DNA polymerase were prepared in rabbits. DNA polymerase-a and -/3 from human lymphoblastic leukemic cells and human placenta were partially purified, the specific activity of each was a b o u t 300 units/mg protein (Dube, D.K. and Seal, G., unpublished results). Homogeneous E. coli DNA polymerase I was purified by the method of Jovin et al. [13] and avian meyloblastosis virus DNA polymerase was purified as described by Kacian et al. [14]. Results and Discussion Crude extracts of T. brucei contain DNA polymerase activity. Analysis of this activity by sucrose density gradient sedimentation reveals one predominant enzyme species using activated DNA as a template (Fig. 1A). The sedimentation rate of the trypanosome DNA polymerase is similar to the larger of t w o DNA polymerases separated from rat liver (Fig. 1B). On the basis of markers, the trypanosome DNA polymerase sediments at 6--8 S which corresponds to that obtained with DNA polymerase-a from a variety of mammalian cells [1--5]. Both the trypanosome DNA polymerase and DNA polymerase-a of rat liver are inhibited by 10 mM N-ethylmaleimide. No smaller species corresponding to DNA polymerase-fl was detected even though homogenation was carried out in high salt (1 M KC1). To preliminarily characterize the DNA polymerase from T. brucei the effect of various chemicals was determined and compared with DNA polymerase-a and -fl from human placenta (Table I). The trypanosome DNA polymerase is entirely inhibited by N-ethylmaleimide at a concentration (20 mM) which also inhibits the mammalian DNA polymerase-a, b u t only partially inhibits DNA polymerase-/3. The response of the trypanosome DNA polymerase to salt is also similar to that observed with mammalian DNA polymerase-a, inhibition being 95% at 0.2 M KC1. In contrast, DNA polymerase-~ activity is unaffected by 0.2 M KC1. In this experiment, ethanol does not adequately distinguish between these DNA polymerases. The resistance of the trypanosome DNA polymerase to Nonidet and Triton-X permits one to use these detergents for preparaing cell-free extracts. The trypanosome DNA polymerase does not appear to be a 7-polymerase (mitochondrial) in that synthesis with 2 pg poly(rA) • oligo(dT) and Mg :÷ or Mn 2÷ is undetectable, less than 5% of that achieved with an equal amount of activated DNA. Synthesis using poly(rA) • oligo(dT) is characteristic of DNA polymerase-7. Our inability to detect a distinct mitochondrial DNA polymerase during the purification is interesting. Trypanosomes have a characteristically large mitochondrion that contains as much as 20% of the total cellular DNA. It has been routinely difficult to extract DNA polymerases from mitochondria in many cells including protozoa. Thus our inability to find a mitochondrial DNA polymerase is not adequate evidence in itself to indicate its absence in trypanosomes. We have recently reported that DNA polymerase-a from a variety of

13

A 0 >,o

1.o 100

~Z~.,

o ......................

Q

Trypanosome DNA Pol.

60

~ o

40

H Z

~

C 20 ¢1) Q..

Q.

0

6

Top

12

10

f8

Fraction Number

Bottom

I 5

I t0

\

~, t5

I 20

Preincubation (min at 45°C)

Fig. 1. S e d i m e n t a t i o n o f D N A p o l y m e r a s e using e x t r a c t s f r o m t r y p a n o s o m e a n d r a t liver. S u p e r n a t a n t f r a c t i o n s w e r e o b t a i n e d f r o m h o m o g e n i z e d t r y p a n o s o m e s a n d r a t liver as i n d i c a t e d in M e t h o d s . T h e supern a t a n t s w e r e c o n c e n t r a t e d b y e v a p o r a t i o n a g a i n s t 50 m M Tris-HCl b u f f e r , p H 7.4, c o n t a i n i n g 5% glycerol and 1 mM dithiothreitol. 0.25-ml aliquots were layered onto 5 ml linear gradients containing 5 to 20% sucrose c o n t a i n i n g 50 m M Tris-HCl, p H 7.4, 0.1 M KC1 a n d 1 m M 2 - m e r c a p t o e t h a n o l . Centrif u g a t i o n was c a r r i e d o u t at 4 ° C for 19 h a t 4 0 0 0 0 r e v . / m i n in a S p i n c o SW 50.1 r o t o r . 0 . 2 5 - m l f r a c t i o n s w e r e c o l l e c t e d f r o m the t o p of t h e t u b e a n d D N A p o l y m e r a s e a c t i v i t y was m e a s u r e d using 0 . 0 4 m l of e a c h f r a c t i o n . A c t i v i t y in the p r e s e n c e of N - e t h y l m a l e i m i d e was d e t e r m i n e d b y p r e i n c u b a t i n g e a c h f r a c t i o n w i t h 10 m M N - e t h y l m a l e i m i d e f o r 3 0 m i n at 4 ° C p r i o r t o m e a s u r i n g D N A p o l y m e r a s e a c t i v i t y . Fig. 2. H e a t i n a c t i v a t i o n of D N A p o l y m e r a s e . T r y p a n o s o m e D N A p o l y m e r a s e was p r e p a r e d as d e s c r i b e d in M e t h o d s . D N A polymerase-c~ a n d -/3 f r o m r a t liver w e r e the p o o l e d a c t i v e f r a c t i o n s f r o m the s u c r o s e g r a d i e n t d e s c r i b e d in Fig. 1. H e a t i n a c t i v a t i o n o f d i f f e r e n t D N A p o l y m e r a s e s was c a r r i e d o u t as follows: for e a c h s a m p l e tested, 0.5 m l a l i q u o t c o n t a i n i n g 0.5 t o 1.0 u n i t s of D N A p o l y m e r a s e was a d j u s t e d to 1 m g / m l of p r o t e i n w i t h b o v i n e s e r u m a l b u m i n , 10 m M Tris-HC1, p H 7.8, a n d 0.01 M KC1. T h e m i x t u r e was i n c u b a t e d at 4 5 ° C . A t the t i m e s i n d i c a t e d 20-~ul a i i q u o t s w e r e r e m o v e d a n d a s s a y e d f o r D N A p o l y m e r a s e a c t i v i t y at 3 7 ° C as d e s c r i b e d in M e t h o d s .

TABLE I E F F E C T O F N - E T H Y L M A L E I M I D E , KC1, E T H A N O L A N D D E T E R G E N T S ON T H E D N A P O L Y M E R ASES FROM T R Y P A N O S O M E S AND H U M A N P L A C E N T A The source g r a d i e n t in cals. 1 0 0 % Polymerase

of t r y p a n o s o m e D N A P o l y m e r a s e w a s t h e c o m b i n e d p e a k f r a c t i o n s [ 1 ~ 1 4 ] o f t h e s u c r o s e Fig. 1A. R e s u l t s are g i v e n as t h e p e r c e n t o f t h e a c t i v i t y in t h e p r e s e n c e of t h e i n d i c a t e d c h e m i c o r r e s p o n d s t o 16 p m o l of t o t a l n u c l e o t i d e i n c o r P o r a t e d in 15 rain, w i t h t h e t r y p a n o s o m e a n d 6 0 P m o l w i t h h u m a n p l a c e n t a D N A p o l y m e r a s e - ~ a n d o/3.

Addition

None N-ethylm aleimid e KC1 (0.3 M) Ethanol (10% w/v) Ethanol (20% w/v0 N o n i d e t P-40 ( 0 . 2 5 % ) N o n i d e t P-40 ( 0 . 5 % ) Triton X-100 (0.5%)

% Activity Trypanosome

Human placenta polymerase-~

Human placenta polymerase-~

100 0--1 7.6 65 11 110 110 102

100 0.5 5 30 3 100 100 100

100 60--90 100 60 20 100 100 100

14 mammalian sources is comparatively resistant to thermal denaturation whereas DNA polymerase-fl is rapidly denatured by preincubation at 45°C [15]. The DNA polymerase-~ and -fl from rat liver exhibits similar differences in thermolability and confirms these findings in another species (Fig. 2). The thermolability of the trypanosome DNA polymerase resembles that of DNA polymerase-~; there is no detectable loss of activity during preincubation at 45°C for 20 min. The effect of two possible inhibitors on the trypanosome DNA polymerase is shown in Table II. Phosphonoacetic acid has been reported to be a specific inhibitor for DNA polymerase induced by infection by Herpes virus [16] and ethidium bromide has been used as a chemotherapeutic agent for treatment of trypanosomiasis [17]. We find that the trypanosome DNA polymerase and the DNA polymerase-~ and -fl from acute lymphatic leukemic cells are only slightly inhibited by phosphonoacetic acid at concentrations as high as 0.1 mM. At a similar concentration, DNA polymerase induced by Herpes virus is inhibited at least 99% [16]. However, at higher concentrations it inhibits both DNA polymerase-~ and -ft. The inhibition of DNA synthesis by ethidium bromide could involve interaction of the ethidium with the DNA template. We find no specificity for this inhibition; DNA polymerases from trypanosomes and human lymphocytes are similarly inhibited. So far, our evidence indicates that the major DNA polymerase in trypanosomes is similar to DNA polymerase-~ in mammalian cells. This polymerase however, can be distinguished from mammalian DNA polymerases on the basis of antigenicity (Table III). DNA polymerases were preincubated with specific antisera in a solution of 0.5 ml containing 100 pg bovine serum albumin and 50 mM Tris-HC1, pH 7.5, for 20 min at 37°C. Afterwards the reaction mixture was centrifuged at 20 000 × g for 30 min at 4°C and the DNA polymerase activity determined on aliquots of 0.02 ml as described in Methods. Reactions were carried out in the presence and absence of the indicated antisera and the percent inhibition was calculated. Purified trypanosome DNA polymerase is not inhibited by antiserum against mammalian DNA polymerase-~ even when this antiserum is added in amounts 100-fold greater than that which would inhibit DNA polymerase-~ from HeLa cells. The same antiserum inhibits DNA

T A B L E II E F F E C T O F SOME I N H I B I T O R S O N D N A P O L Y M E R A S E T h e r e a c t i o n c o n d i t i o n s are the s a m e as given u n d e r M e t h o d s . T h e s o u r c e o f D N A p o l y m e r a s e - ~ and -fl was h u m a n l y m p h o b l a s t i c l e u k e m i c cells. Each assay c o n t a i n e d 0 . 2 units o f D N A p o l y m e r a s e . Addition

None P h o s p h o n o a c e t i c acid ( 1 0 -6 M) P h o s p h o n o a c e t i c acid ( 1 0 -4 M) E t h i d i u m b r o m i d e ( 0 . 5 ~g) E t h i d i u m b r o m i d e ( 1 . 0 pg) E t h i d i u m b r o m i d e (4 ~g) E t h i d i u m b r o m i d e (S }Jg)

% Activity Trypanosome

DNA polymerase-~

DNA polymerase-fl

100 124 83 74 66 19 9

100 I00 84 42 20.8 5.8 1.8

I00 I00 86 95 85 44 15

15 TABLE III EFFECT OF SPECIFIC ANTISERA AGAINST DNA POLYMERASES AMV, avian myeloblastosis virus. Antisera

DNA polymerase

(units)

Activity (%)

A n t i - H e l a DNA polymerase-~ ( I 0 0 pg) A n t i - H e l a DNA polymerase-~ (100 pg)

Trypanosome Trypanosome Hela ALL-~ ALL-~ Trypanosome E. coli Pol I Trypanosome AMV DNA polymerase

(0.02) (0.02) (0.1) (0.1) (0.05) (0.02) (2) (0.20) (2)

110 109 5 5 106 103 4 112 1

Anti-Hela DNA polymerase-~ (40 Dg) Anti-Hela DNA polymerase-~ (40 ~g) Anti-Hela DNA Polymerase-~ (100 pg) Anti-E. coli Pol I (20 pg) Anti-E. coli Pol I (20 pg) Anti-AMV DNA polymerase (20 pg) Anti-AMV DNA polymerase (20 rig)

polymerase-a from acute leukemic lymphocytes but fails to inhibit DNA polymerase-~. This result confirms the DNA polymerase group specificity of antiserum against DNA polymerase-a reported by Spadari et al. [18]. Also, trypanosome DNA polymerase is not inhibited by antisera against DNA polymerases purified from avian myeloblastosis virus and E. coli DNA polymerase I (Pol I). Antisera against human DNA polymerase-a has been shown to inhibit DNA polymerase-a from a variety of mammalian cells [19]. Also, antisera against calf thymus DNA polymerase-a has been shown to inhibit DNA polymerase~ purified from regenerating rat liver. It is unexpected therefore that the trypanosome activity is unaffected by anti-mammalian DNA polymerase-a. These studies indicate that even though trypanosome DNA polymerase shares a host of properties with mammalian DNA polymerase-a, it is antigenically dissimilar. Up to now the only evidence suggesting a possible mechanism of antigenic variation has been the amino acid sequence data of Bridgen [20]. Because only a very small portion of the variant antigen protein was sequenced it would be difficult to make conclusions on the mechanism of antigenic variation from these data. Very recent work showing cross-reacting antigenic determinatants of the variant antigen proteins [21] would suggest that there may be alternative explanations for the great variability of the surface antigens such as somatic rearrangement of genes and even possibly a mutagenic DNA polymerase. These possible explanations have not as yet been considered in the literature and deserve further attention.

Acknowledgements This work has been supported by the National Institutes of Health grants (CA-11524 and CA-12818) to Dr. Lawrence A. Loeb and by grants (CA-06927 and RR-05539) to The Institute for Cancer Research and by an appropriation from the Commonwealth of Pennsylvania. ILRAD Publication No. 16.

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Loeb, L.A. (1974) The Enzymes, X, pp. 173--209, Academic Press, New Y ork Fansler, B.F. (1974) Int. Rev. Cytol. Suppl. 4, 363--415 Holmes, A. and Jo hnston, J. (1975) FEBS Lett. 60, 233--243 Bolden, A., Noy, G. and Weissbach, A. (1977) J. Biol. Chem. 252, 3 3 5 1 - - 3 3 5 6 Chang, L.M.S. (1976) Science 191, 1183--1185 McLannan, A.G. and Kerr, H.M. (1975) Nucl. Acid Res. 2, 223--237 Creran, M. and Pearlman, R.E. (1974) J. Biol. Chem; 249, 3123--3131 Lanham, S.M. and Godfrey, D.G. (1970) Exp. Parasitol. 28, 521--534 Loeb, L.A. (1969) J. Biol. Chem. 244, 1672--1681 Loeb, L.A., Slater, J.P., Ewald, J.L. and Agarwal, S.S. (1971) Biochem. Biophys. Res. Commun. 42, 147--153 Jovin, T.M., Englund, P.E. and Bertch, L.L. (1969) J. Biol. Chem. 244, 2996--3008 Kacian, D.L., Watson, K.F., Burny, A. and Spiegelman, S. (1971) Biochim. Biophys. A c t a 246, 365--383 Dube, D.K., Seal, G. and Loeb, L.A. (1977) Biochem. Biophys. Res. Commun. 76, 483--487 Mao, J.C.H., Robishaw, E.E. and Overby, L.R. (1975) J. Virol. 15, 1281--1283 Williamson, J. (1970) in The African T r y p a n o s o m e s (Mulligan, H.W., ed.), pp. 125--224, George Allen and Unwin, L o n d o n Spadari, S., Muller, R. and Weissbach, A. (1974) J. Biol. Chem. 249, 2991--2992 Chang, L.M.S. and Bollum, F.J. (1972) Science 175, 1116--1117 Bridgen, P.J., Cross, G.A.M. and Bridgen, J. (1976) Nature 263, 613--614 Barbet, A.F. and MeGuire, T.C. (1978) Proc. Natl. Acad. Sci. U.S. 75, 1989--1993