ELSEVIER

Purification

Molecular and Biochemical Parasitology 73 (1995) 53-62

MOLECULAR

f2kHEMIcAL PARASITOLOGY

and characterization of a P-like DNA polymerase from Trypanosoma cruzi

Juan Venegas *, Aldo Solari

Departamento de Bioquimica, Facultad de Medicina, Universidad de Chile. Casilla 70086, Santiago 7, Chile

Received 22 November 1994; accepted 31 May 1995

Abstract

A DNA polymerase was purified to near homogeneity from Trypanosoma cruzi epimastigotes. This preparation had a

major polypeptide of 50 kDa and a minor band of 45 kDa. SDS-PAGE studies and a novel calorimetric activity gel technique demonstrated that the 50-kDa polypeptide chain is the catalytic subunit of this T. cruzi DNA polymerase. Western blot analysis of different purification stage fractions strongly suggests that this 50-kDa protein is the intact catalytic subunit

and does not correspond to a degradation product from a larger one. This T. cruzi DNA polymerase is insensitive to aphidicolin, butylphenyldeoxyguanosine triphosphate, berenil, ethidium bromide and N-ethylmaleimide, but is markedly inhibited by the dideoxythymidine triphosphate analogue. Studies with different DNA templates showed that the DNA polymerase prefers activated DNA as substrate and that it cannot elongate oligoriboadenylate primers. The data presented in

this paper are consistent with the hypothesis that this enzyme corresponds to a p-like DNA polymerase present in the parasitic protozoon T. cruzi.

Keywords: Trypanosoma cruzi; DNA polymerase; DNA polymerase, P-like; Purification

1. Introduction types of DNA polymerases described in higher eu-

In an broad range of eukaryotic organisms five types of DNA polymerases have been described, denominated DNA polymerases (Y, p, y, S and E [l-5]. These enzymes have been poorly studied in algae and protozoa and it is not clear if the same

karyotes are present in these unicellular organisms. Recently, diverse reports have shown that not only an a-like DNA polymerase is present in protozoa

[6-9,101, since a DNA polymerase 6 gen was cloned from Plasmodium falciparum [ll] and a y-like en- zyme was detected in the same protozoon [12].

Abbreviations: BCIP, 5-bromo-4-chloro-3-indolyl phosphate; berenil, 4’,4’-diazoamino-dibenzamidine diaceturate; BSA, bovine serum albumin; BuPdGTP, N2-( p-n-butylphenyl)-dGTP; dNTP, deoxynucleoside triphosphates; ddll?P, 2’,3’-dideoxy-m, DlT,

dithiothreitol; NBT, nitroblue tetrazolium salt.

* Corresponding author. Present address: Lab. Prof. Thto Baltz,

Universitt de Bordeaux II, Bgt 3a, ler etage, Bordeaux. France.

The DNA polymerase p has been thoroughly studied in vertebrates [l-5,13-19]. The general

characteristics of this enzvme are its low molecular + mass (about 40 kDa), its resistance to aphidicolin,

butylphenyl-dGTP and N-ethylmaleimide (NEM), and its sensitivity to dideoxythymidine triphosphate

(dd’ITP). However, studies in Drosophila cells have Tel.: (33-57) 571-014; Fax: (33-57) 571-01:. shown the existence of a DNA polymerase p with a

0166-6851/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0166-6851(94)00091-7

54 J. Venegas, A. Solari/Molecular and Biochemical Parasitology 73 (1995) 53-62

high molecular mass (110 kDa), but with biochemi- cal properties very similar to those described in vertebrates [20].

Several NEM-resistant DNA polymerases have been isolated from lower eukaryotic cells [21-261. These enzymes were only partially purified and in none of them the catalytic polypeptide was identi- fied. Recently a P-like DNA polymerase from the trypanomatid Critidia fasciculata [27,28] was puri- fied to homogeneity. This enzyme was partialy inhib ited by NEM [27]. On the other hand, a probable homolog of mammalian DNA polymerase /3 was purified to homogeneity from the yeast Saccha- romyces cerevisiae [29]. It is very interesting that this enzyme has a molecular mass of 68 kDa and was sensitive to NEM. In order to investigate the structural and biochemical similarities which exist between the protozoan and vertebrate DNA poly- merases, we studied the parasite Trypanosoma cruzi.

double-strand DNA-cellulose were from Sigma. DEAE-cellulose (DE-521 and phosphocellulose (Pll), were from Whatman. DNA and RNA syn- thetic homopolymers were from Pharmacia. Digoxi- genin-11-dUTP and the anti-digoxigenin alkaline phosphatase conjugate were from Boehringer- Mannheim. [ 3 H]dTTP, [ 3H]dATP and [ 3 H]dGTP were from New England Nuclear. BuPdGTP was kindly given by G. Wright (Worcester, MA, USA).

2.2. DNA polymerase assay

Three different assays were used:

2.2.1. Reaction mixture with activated DNA

T. cruzi is the etiological agent of Chagas’ dis- ease, a very important human health problem in Central and South America [30,31]. Nevertheless, to date, there is no efficient drug treatment against this parasitosis [32]. One potential chemotherapeutic tar- get to be investigated in these cells is their DNA polymerases.

In previous studies in our laboratory different DNA polymerase fractions were isolated from T. cruzi parasites, but they were not purified exten- sively and none of them could be classified as the (Y, p, y, 6 or E types described in higher eukaryotes [33,34]. In the present work we show the purification to near homogeneity of a 50-kDa DNA polymerase from T. cruzi epimastigote forms. The physical and biochemical characteristics reported in this paper show that this enzyme corresponds to a /?-like T. cruzi DNA polymerase, analogous to the vertebrate counterparts.

In a final volume of 50 ~1 the assay contained 50 mM Tris . HCl (pH 8.0)/10 mM dithiothreitol (DTT)/lO mM Mg . acetate/100 pg ml-’ BSA/12 PM [3H]TTP (74 cpm pmol-‘)/530 pg ml-’ acti- vated calf thymus DNA/20 ,xM each of dATP, dCTP, dGTP. After incubation for 30 min at 37°C the reaction was stopped by the addition of 2 ml of cold 10% trichloroacetic acid, incubated 20 min at 0-4°C and the acid-insoluble radioactive material collected on glass-fiber discs (Whatman GF/A). The discs were washed with cold 2% trichloroacetic acid, dried and counted in a liquid scintillation counter.

2.2.2. Reaction mixture with polydeoqnucleotide templates

2. Materials and methods

This assay was similar to that described above, but the activated DNA was changed for the synthetic template-primer homopolymers indicated in Table 2. The concentration of these template-primer sub- strates was 0.2:0.08 AZ6a ml-’ in each assay, with the corresponding 12 E_LM f3H]dNTP (40-60 cpm pmol-‘) as complementary labeled substrate. The reaction was incubated 1 h at 28°C and the following procedures were conducted as already described. Template-primer homopolymers were prepared as described [ 151.

2.1. Materials 2.2.3. Reaction mixture with polyadenylic acid tem-

plate (poly(A)) E. coli DNA polymerase I Klenow fragment, In a final volume of 50 ~1 the assay contained 50

aphidicolin, dd’ITP, berenil, DNase I, poly(dC), mM Tris . HCl (pH 8.9)/10 mM MnClJO.1 M anti-rabbit-alkaline phosphatase conjugate, poly(eth- KC1/300 pg ml-’ BSA/0.4:0.2 A,,, poly(A)- ylene glycol) (molecular mass about 6-8 kDa) and :oligo(dT),,_,,/SO PM [3H]dTTP (60 cpm pmol-‘).

J. Venegas, A. Solari / Molecular and Biochemical Parasitology 73 (1995) 53-62 55

The mixture was incubated for 1 h at 28°C and the

remaining procedures were conducted as indicated

before. One unit of DNA polymerase activity was defined

as the amount of the enzyme which catalyzes the incorporation of 1 nmol of the four dNTP into

acid-insoluble material in 1 h at 37 or 28°C when

activated DNA or synthetic homopolymer is used as substrate, respectively.

2.3. Gel electrophoresis

SDS-PAGE was prepared according to the discon-

tinuous system described by Laemmli [35], adapted to Bio-Rad mini-Protean II dual slab cells (7 X 8 X 0.05 cm) with the separating gel containing 7.5%

acrylamide.

2.4. Activity gel analysis

Calorimetric detection of DNA polymerase activ- ity after SDS-PAGE was performed as was previ- ously described [36].

2.5. DNA activation

Calf thymus DNA was activated by DNase I treatment as previously described [37].

2.6. Parasites

The proliferative T. cruzi epimastigote forms (clone Dm-28) were cultured in a modified LIT medium [38] at 28°C with gentle shaking to the exponential phase (about 5 X 10’ parasites ml-‘). The cells were collected by centrifugation at 5000 X g for 10 min, TinSed twice with PBS buffer containing

50 mM sodium phosphate (pH 7.2) and 30 mM NaCl. The parasites were then stored at - 80°C until they were used to purify the DNA polymerase.

2.7. Purification of DNA polymerase (Y from Xeno- pus laevis ovaries

DNA polymerase CY was partially purified from ovaries (26 g) of two frogs essentially as described [17] but with some modifications, such as the elution of the first DEAE-cellulose in one step and the

addition of a further purification stage using a

DNA-cellulose column.

2.8. Production of antibodies

An antiserum against the low-molecular-mass DNA polymerase was obtained by immunization of a

rabbit with a preparation of highly purified enzyme equivalent to the fraction V shown in this paper. All the inoculations were done by intramuscular injec-

tions in the thigh muscle of a rear leg. The first inoculum was about 10 pg of sample with Freund’s

complete adjuvant. After one month, two successive new doses with about 5 pg of that protein without

any adjuvant were injected in the rabbit, at one month intervals. Two weeks after the third inocula-

tion the rabbit was bled and the collected blood was used to obtain the antiserum against the T. cruzi DNA polymerase used in this work.

2.9. Western blot analysis

Western blots were performed according to the technique described elsewhere [39], using a mini-gel system and the semi-dry electroblotting system

(Sartoblot II-S Sartorius) for transferring the proteins to nitrocellulose filters. The rabbit antiserum and the antirabbit alkaline phosphatase conjugate, were used

at a dilution of 1:200 and 1:5000, respectively. India Ink staining of total protein transferred to

the nitrocellulose filters was performed as described

[401.

2.10. Protein determination

The protein concentration was determined accord- ing to Bradford [41].

2.11. Purification of a low-molecular-mass DNA polymerase from T. cruzi epimastigote forms

All procedures were conducted at 0-4°C. Epi- mastigotes (20 g, about 2.6 X 10” parasites) were

suspended in 60 ml of lysis buffer (50 mM Tris . HCl (pH 8.0)/2 mM 2-mercaptoethanol/O.l mM EDTA/l mM Ncu-p-tosyl-L-lysine chloromethylke-

tone/6.3 pg ml-’ leupeptin/lO mM NaHSO,/l E.LM pepstatin/l mM phenylmethylsulfonyl fluo-

56 J. Venegas, A. Solari /Molecular and Biochemical Parasitology 73 (1995) 53-62

ride/l% Nonidet P-401, homogenized with a Dounce

homogenizer and centrifuged (10000 X g for 15

min). The supernatant extract (Fraction I) was loaded onto a phosphocellulose column (3.5 x 14 cm) equi- librated with a buffer A (50 mM Tris . HCl

(pH 8.0)/2 mM 2-mercaptoethanol/O. 1 mM

EDTA/lO% (v/v> glycerol/0.5% poly(ethylene glycol)/O.l mM No-p-tosyl+lysine chlorometh- ylketone/l pug ml- ’ leupeptin/lO mM

NaHSOJ0.2 PM pepstatin/l mM phenylmethyl- sulfonyl fluoride/50 mM KCl). The column was

washed with three volumes of buffer A containing

0.1 M KC1 and eluted with buffer A containing 0.2 M KCI. DNA polymerase activity eluted in this

fraction (320 ml> was precipitated by slow addition

of (NH,),SO, to 80% saturation. The precipitate was collected by centrifugation, dissolved in 60 ml of buffer A, centrifuged at 10000 X g for 10 min

and the supernatant loaded onto a Sephadex G-25 column (3.2 X 26 cm) equilibrated in buffer A. Ac- tive fractions with the same conductivity of buffer A were pooled (Fraction II). Fraction II was loaded

onto a DEAE-cellulose column (2.5 X 8 cm) equili-

brated with buffer B (= buffer A + 20% glycerol). The flowthrough fractions with major activity were

pooled (Fraction III) and loaded onto a double strand

DNA-cellulose column (1.5 X 6 cm) equilibrated in buffer B. The column was washed with 20 volumes of the above buffer and eluted with 10 volumes of a 50-600 mM KC1 gradient in buffer B. The fractions

with DNA polymerase activity were pooled (Fraction IV), and dialyzed against buffer B. Fraction IV was

loaded onto a second phosphocellulose column (0.35 X 5 cm) equilibrated in buffer B. The column was

washed with ten volumes of buffer B and eluted with

10 volumes of a 50-600 mM KC1 gradient in the

above buffer. The fractions with DNA polymerase

activity were pooled (Fraction V), dialyzed against buffer B containing 50% (v/v> glycerol, in which the poly(ethylene glycol) was omitted. This dialyzed fraction V was aliquoted and stored at -20°C for several months without significant loss of activity.

3. Results and Discussion

With this purification scheme a DNA polymerase preparation with a specific activity of 6705 units

-r and an estimate yield of 28.8% was obtained gble 1). The principal differences of this protocol

compared with others used before [33,34] was the parasite disruption with a low-ionic-strength buffer and the inmediate chromatography onto a phospho- cellulose column. This is a critical step, since there- after the enzyme activity remained stable and a

better yield was obtained. It is worth mentioning that the elution of the first phosphocellulose column with 0.2 M KC1 separated the total DNA polymerase

activity in an eluted and a bound fraction. The further purification of the eluted fraction is described

in this paper.

3.1. SDS-PAGE analysis of different purification fractions

Aliquots from each purification step of the T. cruzi DNA polymerase were analyzed by SDS-PAGE

(Fig. 1). The el ec rophoretic pattern shows that the t highest purified fraction has a major protein band of

50 kDa and a minor one of 45 kDa (lane 4).

Table 1

Purification of p-like DNA polymerase from Trypanosoma crzui epimastigote forms

Fraction Step Volume Protein Total

(ml) (mg) activity a

(units)

Specific

activity

(units mg-’ 1

Yield

(o/o)

I Extract 60 654 1224

II 1st Phosphocellulose 70 29.7 2212

III DEAF+cellulose 66 9.0 2746

IV DNA-cellulose 12.0 0.216 1344

V 2nd Phosphocellulose 2.5 0.095 637

a All the assays were conducted using activated DNA as described in Materials and methods.

b ND, not determined.

1.87 NDb

74.5 100

305 124

6222 61

6705 28.8

J. Venegas, A. Solari/Molecular and Biochemical Parasitology 73 (1995) 53-62

kDa M 1 2 3 4

:E 924

45

29

Fig. 1. SDS-PAGE analysis of fractions from different purification

steps of low-molecular-mass T. cruzi DNA polymerase. The gel was stained with Coomassie blue and the samples were: lane 1,

Fraction II (2.1 pg); lane 2, Fraction III (0.8 pg); lane 3, Fraction

IV (0.2 yg); lane 4, Fraction V (0.4 pg). The samples and the

procedures were described in Materials and methods. Lane M,

molecular mass markers: carbonic anhydrase (29 kDa), ovalbumin

(45 kDa), BSA (66 kDa), phosphorylase b (97.4 kDa), P-galacto-

sidase (116 kDa) and myosin (205 kDa).

Several efforts were made to obtain a homoge-

neous preparation with a single protein, using more purification steps after the last phosphocellulose col- umn shown here, but only we obtained a less active fraction with the same two polypeptide chains. It is possible that the 45-kDa protein corresponds to a

degradation fragment from the 50-kDa species, but further studies are required to prove that idea.

3.2. Identification of the T. cruzi DNA polymerase catalytic polypeptide

Aliquots of DNA polymerase fractions eluted from the DNA-cellulose column were analyzed by SDS- PAGE (Fig. 2). Fig. 2 shows a clear correlation between the 50-kDa polypeptide and DNA poly- merase activity. The 45-kDa protein band is more tightly bound to the DNA-cellulose column and elutes after the DNA polymerase peak (lane 16).

To obtain direct evidence that this 50-kDa polypeptide chain is the T. cruzi DNA polymerase, enzyme fractions from different purification steps were studied by a calorimetric activity gel technique

kDo M IO ,I 12 13 14 15 I6 17 20

205 - 116 - 97.41 66 '

k Fraction Number

Fig. 2. Correlation between DNA polymerase activity eluted from

a DNA-cellulose column and SDS-PAGE analysis. A sample of 5

~1 from each DNA-cellulose fraction (B) was analysed by SDS-

PAGE and stained with Coomassie blue (A) as described in

Materials and methods. The lane numbers in (A) correspond to the

fractions indicated in the graph (B). Lane M, molecular mass

markers.

[36]. Fig. 3 shows that in the most purified fractions

only one DNA polymerase activity band was tected corresponding to the 50-kDa polypeptide.

kDa 0 I 2 3 4 5

de-

Fig. 3. Identification of the T. cruzi DNA polymerase catalytic

polypeptide by activity gel analysis. Lanes l-4 were analysed by

a calorimetric technique which detects DNA polymerase activity

after SDS-PAGE, as described in Materials and methods. Lane 1,

Fraction II (2.1 &; lane 2, Fraction III (0.8 pg); lane 3, Fraction

IV (0.2 Kg); lanes 0 and 4, Fraction V (0.4 pg); lane 5, Klenow

fragment (2 ng). Lane 0 was stained with Coomassie blue.

58 J. Venegas, A. Solari/Molecular and Biochemical Parasitology 73 (1995) 53-62

3.3. Western blot analysis of T. cruzi DNA poly- merase purification

The same purification fractions shown in Fig. 1 were studied by Western-blot technique (Fig. 4). The samples were analyzed with a rabbit anti-serum elicited against a highly purified low molecular mass T. cruzi DNA polymerase preparation and detected with an anti-rabbit secondary antibody conjugated to alkaline phosphatase. The results show that the anti- serum presented a strong reaction with the 50-kDa DNA polymerase band and a weak, but significant reaction, with the minor 45-kDa band (lane 41, which is not detected in the less purified fractions (lanes 1 and 2). None of the purified fractions analysed showed any cross-reacting higher-molecular-mass antigens. Similar results were obtained using a crude extract of T. cruzi epimastigote cells (data not shown). Taken together, these results strongly sup- port the idea that the 50-kDa polypeptide chain is the intact low-molecular-mass T. cruzi DNA poly- merase.

3.4. Characterization of low-molecular-mass T. cruzi DNA polymerase with inhibitors

The effect of some classical DNA polymerase

kDa 012345

Fig. 4. Western blot analysis of fractions from different purifica-

tion steps of the low-molecular-mass T. crtui DNA polymerase. A

single gel was analysed by the Western blot technique using an

antiserum against the p-like DNA polymerase from T. cruzi (lanes l-4), a nonimmune serum (5) and India ink staining (lane

0) as described in Materials and methods. Lane 1, Fraction II (2.1

pg); lane 2, Fraction III (0.8 /.~p); lane 3, Fraction N (0.2 @g)

and lanes 0, 4 and 5, Fraction V (0.4 pg).

4 . 0 A 0 """','I

[N:o,l.,M z"

50 100

[ddTTPJ.pM

Fig. 5. Effect of inhibitors on the enzymatic activity of the T.

cruzi DNA polymerase. Each assay corresponds about to 0.5 units

of Fraction V (see Table 1) from T. cruzi epimastigote (0) and

0.5 units of DNA polymerase cr from X. laevis ovaries (0 1, used

as control enzyme. All the assays were performed with activated

DNA with the following exceptions indicated for each case: (top

left) aphidicolin experiment, assay with 5 PM dClP and 4%

DMSO from the aphidicolin sample; (top right) BuPdGTP experi-

ment, assay with 10 PM dGTP; (middle left) berenil experiment;

(middle right) ethidium bromide experiment; (bottom left) NEM

experiment, assay without Dm, (bottom right) ddTIP experi-

ment, assay with 10 /.LM dTI’P. The assays were pre-incubated

with the corresponding inhibitor at 4°C for 30 min before starting

the enzyme reaction. The assay conditions and the purification of

both enzymes were described in Materials and methods.

inhibitors was examined against the T. cruzi enzyme using a partially purified fraction of DNA poly- merase (Y from Xenopus laevis ovaries as control (Fig. 5). Aphidicolin, a classical inhibitor of verte- brate DNA polymerases (Y, 6 and E [3-51, has no effect on the T. cruzi enzyme, whereas the X. laevis enzyme was inhibited to comparable levels as de- scribed in the literature for a similar preparation [ 171. Analogous results were observed using the specific DNA polymerase (Y inhibitor BuPdGTP [3-51.

J. Venegas, A. Solari/Molecular and Biochemical Parasitology 73 (1995) 53-62 59

The DNA intercalating agent ethidium bromide is used to identify the DNA polymerase y, because this enzyme is highly sensitive to this drug [1,42]. For instance, the typical vertebrate DNA polymerase y is inhibited almost completely by 20 PM of this drug [42]. At this drug concentration the T. cruzi enzyme was inhibited only about 25% (Fig. 5).

Another DNA intercalating agent used in this analysis was berenil. Earlier in vivo studies on T. brucei showed that this drug strongly inhibited mito- chondrial DNA synthesis in this parasite [43]. The

results show that the inhibition of the T. cruzi DNA polymerase did not reach 20% in any of the drug concentrations used.

reagent [l-5]. ddTTP was the strongest inhibitor of

the T. cruzi DNA polymerase (Fig. 5). Concentra- tions as low as 20 PM, corresponding to a dTTP/ddTTP ratio of 1:2, inhibited more than half

of the original enzyme activity. In contrast, at the same drug concentration the _X. laevis DNA poly- merase only was inhibited about 10%. However, the

T. cruzi enzyme was not so sensitive in comparison

to its mammalian counterpart in which a dlTP/ddlTP ratio of 1 could completely inhibit

that enzyme [l]. In general, the inhibition characteristics of the T.

cruzi enzyme are in agreement with the properties described for vertebrate DNA polymerase p [l-5]

N-ethylmaleimide (NEM) is one of the most im-

portant criterias used to identify the vertebrate DNA polymerase /3 because this enzyme is the only one resistant to this drug [l-5]. It has been found that

with 10 mM NEM and in the absence of sulfhydryl

reagents the vertebrate DNA polymerase p con-

serves, at least, the 65% of its total activity [44]. Fig. 5 shows that at 10 mM NEM the T. cruzi DNA polymerase conserved more than 50% of its activity, and even at 20 mM of the drug the enzyme activity

was not inferior to 40%. In contrast, low concentra- tions of NEM strongly inhibit the X. laevis DNA polymerase CY, in agreement with previous reports

n7l.

3.5. Utilization of DNA templates

ddTTP also is using to discriminate between ver- tebrate DNA polymerases /3 and the other types because this enzyme is the most sensitive to that

Similar to that described for some vertebrated DNA polymerase p species [1,3,45], the best tem-

plate for the T. cruzi DNA polymerase was the

gapped calf thymus DNA (activated DNA), regard- less of the covalent metal used as cofactor (Table 2).

Nevertheless, the utilization of this template strongly depends on the optimal gapped conditions of the natural DNA and it is not an easily comparable

parameter between DNA polymerases /3, because several reports show that synthetic deoxypolymers

such as poly(dA-dT) or poly(dA) are better sub- strates than activated DNA [44,46]. It is important to point out that with neither Mn*+ nor Mg2+ any T. cruzi DNA polymerase activity was detected when

Table 2

Utilization of template-primers by a p-like DNA polymerase from Trypanosoma cruzi and a DNA polymerase (I from Xenopus laeuis

ovaries

Template Primer L3H]dNTP DNA polymerase activity a

(pmol h-r)

P-like (Y

Mg2+ Mna+ Mg2+ Mn2+

Activated DNA _ d’lTP 285 332 928 159

Poly(dA) GligddT),, -ts dTl-P 5.9 52 117 56

Poly(A) 01igddT)t2_,s dTTl= ND b 6.3 ND 40

Poly(dT) 01ig0(A),,_,, dATP 0 0 357 400

Poly(dT) OligdW, dATP 12.5 21 0 54

Poly(dC) OligddG),,_,s dGTP 56 28 82 120

a The assays were conducted using about 0.3 and 0.9 units of T. cruzi (Fraction V) and X. laeuis DNA polymerase sample, respectively, as

described in Materials and methods.

b ND, not determined.

60 J. Venegas, A. Solari/Molecular and Biochemical Parasitology 73 (1995) 53-62

poly(dT)-oligo(A) was used as subtrate, while the X. laevis enzyme reached elevated enzyme activities. Furthermore, the results indicated that this difference between the enzymes is due to the nature of the primer, because when the same template was primed with the deoxynucleotide homologue, oligo(dA), the T. cruzi enzyme had significant activity. This result is in agreement with the behaviuor described for vertebrate DNA polymerase p [ 1,2,47,48].

In general, most of the vertebrate DNA poly- merases /3 can use poly(A) [1,2,15,45,46,49,50]. Poly(A)-oligo(dT) was a very poor substrate for the T. cruzi DNA polymerase (Table 2). This result was not due to the primer oligo(dT), because the same primer, annealed with the deoxypolymer poly(dA), was used efficiently by the enzyme. This observation indicates that, under these conditions, it was the ribopolymer template poly(A) which was a very poor substrate for this enzyme. This situation was not due to problems with the ribopolymer template (degrada- tion or bad annealing with the primer), because the preparation of X. laevis DNA polymerase (Y had significant activity using the same assay.

Most of the studied characteristics of the T. cruzi DNA polymerase in this work are in agreement with the properties described for the vertebrate /3 type counterpart. It is clear that this T. cruzi enzyme could not correspond to a (Y, 6 or E type, consider- ing its resistance to aphidicolin, BuPdGTP and its low molecular mass, but also there are strong evi- dences against a possible y type. In this regard we could take into account not only the higher molecu- lar mass of the y species [3-51, but also the strong sensitivity to NEM [3-51, the preference for poly(A) substrates [l] and the sensitivity to ethidium bromide [42]. None of these characteristics were displayed by the T. cruzi enzyme. For this reason the data strongly supports the idea that this low molecular mass T. cruzi enzyme corresponds to a P-like DNA poly- merase presents in this parasite.

Comparing this T. cruzi enzyme with the most purified and characterized probable P-like DNA polymerases isolated from lower eukaryotic cells corresponding to C. fasciculata [27,28] and 5. cere- visiae [29], we could see that the C. fasciculata enzyme is the most similar considering its sensitivity to NEM (50% activity to 2 mM NEM) and its molecular mass. In contrast the 5. cereuisiae enzyme

was inhibited by that drug and has a molecular mass of 68 kDa 1291. However, other biochemical proper- ties are very similar between the T. cruzi and the S. cerevisiae enzymes. For instance, both of them pre- fer activated DNA, as substrate and neither of them could efficiently use poly(A)-oligo(dT), as primer- template. Besides, the sensitivity to ddTTP in both enzymes was very similar and far from that de- scribed for mammalian DNA polymerase p [1,3-51. These results are very interesting because they showed that indeed there are important differences between unicellular P-like DNA polymerases and its mammalian counterpart which could be exploited, in the case of parasite enzymes, as chemotherapeutic target.

New information about this field will arise from the amino-acid sequence and cloning of this T. crud DNA polymerase. To achieve these objectives our laboratory is dedicating its major efforts.

Acknowledgements

We thank to Dr. Lafayette Eaton and Dr. Cather- ine Connelly for critical reading of the manuscript. We thank Dr. Marta Gajardo and Dr. Gittith Sanchez for their invaluable technical assistance in the im- munological methods used in this work. This work was supported by a grant from the UNDP/WORLD BANK/WHO, project ID 890308; a grant from FONDECYT, project 2930021 and DTI from the University of Chile. This work represents the partial fulfillment of the Ph.D. thesis of J.V.

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