FEMS MicrobiologyLetters 95 (1992) 71-76 ,~'>1992 Federation of European Microbiological~)cieties 037S-11)97/92/$1)5.110 Published by Elsevier
FEMSLE I)4975
The D N A polymerases of Leishmania mexicana L i n d a L. N o l a n
t
a n d J o s e H. R i v e r a
Uni~'ersity of Massachusetts, &'hoolof Public Ih'alth. .4mh('r~t. MA, USA
Received 311September 1991 Revision received5 May 1092 Accepted 6 May 1092 Key words: D N A polymerase; Leishmanm mextcana: Chemotherapy
1. S U M M A R Y Two previously isolated D N A polymerases from the parasitic protozoan Leishmania mexicana were further characterized by exposure to inhibitors of mammalian D N A polymerases. D N A polymerase A, a high molecular mass enzyme, and D N A polymerase B, a /3-like D N A polymerase were compared to each other and to their mammalian counterparts regarding pH optimum, utilization of templates, and response to various inhibitors and ionic strengths. The results suggest that the D N A polymerases from L. mexicana differ from the host enzymes and may offer a target for chemotherapeutic intervention.
2. I N T R O D U C T I O N Five classes of D N A polymerase (a, /3, y, (~ and e) have been isolated from higher eukaryotic cells [1-4] and a , / 3 and y-like polymerases from t Correspondence to: Present address: L. Nolan. School of
Public Health, Morrill Science Center, Universityof Massachusetts, Amherst, MA 01IX)3,USA.
parasitic protozoa [5-14]. D N A polymcrases a, and E are nuclear enzymes associated with chromosomal replication: /3 is a low molecular mass nuclear enzyme involved in D N A repair [2,15,16], and y which has been isolated from mitochondria is believed to be responsible for mitochondrial D N A replication [2,4,17]. We have been studying D N A replication in the kinetoplast parasite Leishmania mexicana and have begun studies to characterize the major polymerase activities in these parasites for the purpose of comparing them to host polymerases, particularly a and/3. Although North and Wyler [18] reported studies of in vivo D N A replication of Leishmania parasites, this laboratory is the first to report the isolation and characterization of the leishmaniai D N A polymerases in vitro [68]. Others have described purification of a-like, /3-like [9] and y-like polymerases [14] from the parasitic protozoans Crithidia fasciculata and an a-like polymerase from Trypanosoma bmcei [10] and Trypanosoma cmzi [11]. The purpose of this study is to compare the major D N A polymerase activities (A and B) isolated from Leishmania mericana to a and /3 polymerases isolated from other sources.
72 3. MATERIALS AND METHODS
3.1. Test organism Leishmania mexicana amazonensis (Walter Reed strain 227) obtained from the Leishmania section of the Walter Reed Army Institute of Research were grown in brain-heart infusion media as previously described [6]. 3.2. Preparation of compounds Aphidicolin (Sigma, St. Louis, MO) was prepared in dimethyisulfoxide (DMSO) as a 5 mM stock and diluted with water so that the final concentration of DMSO in the assay was no more than 0.16% (v/v). Suramin, purchased from Miles Pharmaceuticals (West Haven, CT), was made into a 70 mM stock solution in 10 mM Tris, pH 7.5. Further dilutions were made with the same buffer. Butylphenyl dGTP (BuPdGTP), carbonyldiphosphonate (COMDP) and the phosphonoacetic acid derivatives BrPAA, CIPAA, FPAA and F2PAA were generous gifts from Dr. G. Wright, University of Massachusetts Medical Center (Worcester, MA), and were prepared in aqueous solution at the appropriate concentrations. All other chemicals were purchased from Sigma. 3.3. Isolation of DNA polymerases DNA polymerase A, a high molecular mass DNA polymerase sensitive to N-ethylmaleimide (NEM), was isolated from L. mexicana promastigotes as previously described [7]. A low molecular mass DNA polymerase classified as a /3-like enzyme was isolated from promastigotes as previously described [8]. This enzyme will be referred to as DNA polymerase B, to distinguish it from mammalian enzymes and to follow the designation used by Holmes et al. [9] for the Crithidia fasciculata DNA polymerases. 3.4. Drug assays The inhibitory properties of several compounds were determined by pre-incubating the enzyme and drug in the assay mix. In order to characterize the enzymes, selective inhibitors of mammalian DNA polymerases were tested against both enzymes. DNA polymerase A was assayed at
35°C as described [7]. DNA polymerase B activity was measured at 35°C as previously described [8].
4. RESULTS AND DISCUSSION
4.1. Isolation of DNA polymerases Two types of DNA polymerase activity were separated using affinity chromatography with denatured DNA cellulose. The two enzyme activities were designated as DNA polymerase A [7] and a /3-like DNA polymerase [8] according to their molecular mass, pH optimum and response to N-ethylmaleimide. The B enzyme (pol B) was less stable than the DNA polymerase A (pol A) at all stages of the purification. The use of a mixture of protease inhibitors as well as glycerol during the isolation procedures was essential for stability of the DNA polymerases. In addition, the use of a freshly prepared assay mix was critical in obtaining pol B enzyme activity. Difficulty in detecting a low molecular mass DNA polymerase in parasitic protozoans has resulted in conflicting reports from some groups regarding the presence of a low molecular mass DNA polymerase in Trypanosoma brucei [5,10]. In addition, studies on T. cruzi detected only one DNA polymerase of high molecular mass and no/I-like enzymes [11]. Proper characterization of the DNA polymerases from L. mexicana is essential in order to compare them with the host enzymes, a first step in a strategy to develop chemotherapeutic agents. To date, all enzymes isolated from parasitic protozoans have been foand to share some, but not all, of the characteristics of the mammalian enzymes [5,7-14]. 4.2. Characterization studie~ Pol A was slightly stimulated by NaCI or KCI at concentrations of less than 15 mM, but rapidly inactivated by higher concentrations of salt [7]. DNA polymerase B was slightly stimulated by 5 mM KCI only, but was more resistant to inactivation by higher concentrations of NaCI or KCI, with 35% of the activity remaining in the presence of 200 mM NaCI and 43% of the activity remaining in the presence of 200 mM KCi [8].
Table I T e m p l a t e specificity of the L. mexicana D N A polymerases A and B Template-Primer
Labelled nucleotide
Divalent cation
Activated D N A
dTTP dTTP dTTP dTTP dTTP dTTP dGTP dGTP dGTP dGTP dGTP dGTP
Mg 2 " Mn 2 ~ M g 2" Mn 2" M g 2" Mn 2 ÷ M g 2" Mn 2" Mg 2+ Mn 2 ~ M g 2" Mn 2 +
poly(rA).oligo (dT)lo I~fly(dA)" °lig°~dT);2- t~ Activated D N A poly(rC).oligo(dG) m poly(dC)- oligo(dG )t 2_ I~
c~ of activity relative to activated D N A with [ methyl- 311]dTlrP D N A POl A
D N A pol B
100 18 3 31 q9.2 27.5 1.76 11.4 (I 1.3 57.8 5.4
100 0 I).9 0.9 149 0 17 0 2 It 650 0.9
Concentrations of divalent cations were 8 m M MgCI 2 or 11.5 m M MgCI 2.1Adapted from Nolan et al. [7] and Nolan and Rivera [8].)
Table 2 IC50 values of several D N A polymerase inhibitors on the L. mt:ricana pol A and pol B [7,8] Inhibitor
DNA polyme,lse
L. mexicana
Mammalian a (~M) AraCTP Aphidicolin NEM BuPdGTP ddTTP PAA FPAA BrPAA CIPAA FzPAA COMDP Suramin Hemin
- ,, '~ < 100 0.3 R 71-7111 ¢ 20 1611 121) 300 a 300 -
/3
"),
- " R > 111 m M _ b 2 R
R < 11111 > 11111 11.115 71-7111 ¢
8
2 < 101111 > 11) R 2 2t} 70 21Xl" 411 -
~
15 > 11)
21111 ~
A (/zMI
B
R R < 1 I11X1 I1~) R 1511t) 1311 R R R 151) 8 90
> 51111 ~.M R R 5.4 7.5 R R R R R 21111 3 611
T h e IC5. values for the m a m m a l i a n D N A polymerases indicate representative data obtained from published values in the literature [2.19,21,22,25]. Enzymes found to be resistant to inhibition were m a r k e d as R. a It has been reported that, as a rule. m a m m a l i a n polymerase/3 is less sensitive than polymerase a with K i values ranging from 2 - 4 ,aM for polymerase a versus 13-32 t~M for polymerase/3 [2]. , T o 6fir knowledge there is no published data on the inhibition of polymerase/3 by B u P d G T P . However. it has been reported that p o l y m e r a s e / 3 is weakly inhibited by B u P d G T P [24]. ¢ Concentration range at which D N A polymerases a and y are reportedly inhibited by P A A [2]. d Values obtained using poly(dA).oligo~dT) I_,_ is as the template [21]. " T h e s a m e study reported 93% inhibition of D N A polymerase 8 from H e L a cells by 15 ~,M C O M D F using poly(dA)-oligo(dT) IZ%
74 Mammalian D N A polymerase a is inhibited by high ( > 100 raM) concentrations of salt, whereas D N A polymerases /3 and ,5 are stimulated by such concentrations [2]. The optimum pH of the pol A enzyme is mildly acidic to neutral at 6.7, whereas the optimum pH of the pol B enzyme is basic, at 9.0. Table I shows a comparison of the activity of the enzymes with several template-primers. The pol A had a template preference for activated D N A and used poly(dA), oligo(dT)t,_ i~ equally as well, with only 6(1% of the activity when poly(dC). oligo(dT)lz_18 was the template [7]. Pol B showed a six-fold preference for poly(dC), oligo(dT)tz_18 as the template over activated D N A [8]. The preferred template for D N A polymerases a and 13 is activated DNA, whereas the mitochondrial D N A polymerase is more active with poly(rA). oligo(dT). A notable point is the inability of pol B to utilize Mn ÷z as the divalent cation activator. In contrast, mammalian D N A polymerase /3 is capable of using both Mn 2÷ and Mg -'+ [2]. 4.3. hthibitor studies Exposure of the enzymes {o specific D N A poly~crase inhibitors showed the L. mexicana enzymes t,, be different from one another and from mammalian enzymes in their sensitivity to various compounds (Table 2). Both enzymes were resistant to aphidicglin, a mammalian D N A polymerases a, ~ and E inhibitor. The response of these enzymes to the mammalian D N A polymerase a inhibitor BuPdGTP was interesting, in the presence of 100 # M dGTP, pol B was twenty-fold more sensitive to this compound than pol A with a concentration that inhibits activity by 5(1% (IC~(,) of 5.4 p,M, whereas the pol A was inhibited with an IC50 of 100 # M . Phosphonoacetic acid (PAA) was a weak inhibitor of the pol A with only 35% inhibition at 2 raM. Pol B was resistant to PAA at concentrations of up to 2 mM. Mammalian /3 polymerase has been found to be resistant to inhibition by this compound [2]. Several PAA analogues [21] were tested against the L. mexicana D N A polymerases (Table 2). Pol B was completely resistant to inhibition by the fluoro, bromo, chloro, and difluoro analogues of PAA (FPAA, BrPAA, C!PAA, F2PAA, respec-
tively). Pol A was resistant to BrPAA, CIPAA, and F2PAA. FPAA, a monohalogenated derivative of PAA, inhibited pol A with an IC5, of 131) p,M, resulting in a greater than ten-fold increase in inhibition compared to PAA. F P A A also exhibited potent inhibition of the calf thymus D N A polymerases a and `5 (Table 2; ref. 21). COMDP, a specific inhibitor of mammalian D N A polymerase ,5 (refs. 15, 22; G. Wright, personal communication), was inhibitory to both enzymes from L. mexicana. The pol A enzyme was more sensitive to C O M D P than the pol B with ICs0s of 150 and 2011/xM, respectively (Table 2). The response of these L. mexicana enzymes to non-specific inhibitors showed the unique properties of each enzyme (Table 2). Hemin, a critical nutritional component of the leishmanial growth media [6], was found to inhibit both enzymes, inhibiting pol B with an ICs~j of 6(I # M versus an ICs~ of 91) p~M for the pol A. Heroin inhibits D N A synthesis reversibly by binding D N A polymerase and causing it to dissociate from the template [23]. Suramin, a drug used in the treatment of trypanosomiasis that has also been found to be a strong competitive inhibitor of the reverse transcriptase of a number of animal retroviruses [24], was found to be a potent inhibitor of the 1.. mexicana D N A polymerases. Suramin gave an IC5~ of 8 # M for pol A and 3 # M for pol B (Table 2). Our characterization studies have shown the L. mexicana pol A and pol B to differ from each other in molecular mass, pH optimum, template specificity, and response to salt and inhibitors. In addition, our studies have shown that pol A and pol B share similar properties such as pH optimum, molecular mass, and sensitivity to specific inhibitors such as NEM, with their mammalian counterparts. The assignment of poi A to a specific class among the eukaryotic D N A polymerases is made difficult by its utilization of template (Table 1) and by the particular response of this enzyme to inhibitors (Table 2). Although this high molecular mass enzyme shows a-like properties such as inhibition by N E M and salt, insensitivity to ddTTP, and preference for Mg 2-~ and activated DNA, it also displays characteristics that do not fit the a type. Pol A also shows
characteristics of the ~5 type, such as resistance to aphidicolin and utilization ( a l t h o u g h at low levels; Table 1) of ribonucleotide t e m p l a t e w h e n Mn-' is the divalent cation. O n the o t h e r hand, the low sensitivity to B u P d G T P and the s o m e w h a t high sensitivity to C O M D P point toward characteristics o f the D N A p o l y m e r a s e s ~ and ~ [19]. Pol B can be m o r e easily classified as a /3-like enzyme b a s e d on its low molecular mass, resistance to N E M , a n d sensitivity to d d T T P . However, pol B failed to cross-react with an anti-rec o m b i n a n t m o u s e D N A p o l y m e r a s e /3 a n t i s e r u m using e n z y m e neutralization studies [8]. Using e n z y m e neutralization studies, as well as i m m u n odiffusion and i m m u n o e l e c t r o p h o r e s i s , C h a n g and Bollum [25] s h o w e d that 7: bnu'ei D N A p o l y m c r a s e /J did not cross-react with an antis e r u m against calf t h y m u s D N A p o l y m e r a s e ft. O b s e r v a t i o n s of differences with the m a m malian p o l y m e r a s e have b e e n m a d e on the enzymes o f o t h e r p r o t o z o a n s [7-14,20,25], suggesting that D N A replication in h i g h e r e u k a r y o t e s a n d p r o t o z o a n s may differ. Such differences are being characterized in this laboratory in the s e a r c h lor potential anti-parasitic agents.
ACKNOWLEDGEMENTS W e are grateful to Drs. N a s e e m a Khan, G e o r g e Wright and Lech Dudycz for giving us advice and p o l y m e r a s e inhibitors to test. We also wish to thank Therese Lapointe for typing the m a n u s c r i p t . This w o r k was s u p p o r t e d by a contract f r o m the U.S. A r m y Medical R e s e a r c h a n d D e v e l o p m e n t C o m m a n d , D A M D 17-87-C-7146, to L.L.N.
REFERENCES [I] Sctwassi. A.I.. Plevani. P. and Bertazmni. U. 119811) Trends Biochem. Sci 5. 335-337.
[2] FrT'. M. and t.~b. L.A. (1985) Animal Cell DNA Imlymerases. ('RC Press. I~ca Raton, FL. [3] Loeb. L.A. (Itl741 In: The En~'mes. IP~yer. P.D.. Ed.). pp. 174-2119. Academic Press, Nev."York. [4] Wang, T.S.F. (1~91) Annu. Re,;. Biochem. ~11,513-552. [5] Chang, L.M.S.. Cheriatundam. E.. Mahone.v, E.M. and Ccrami. A. (1981)) Sclence 2118.510-511. [6] Nolan. L.L. I It'S71 Antimicrob. Agents C'hemotcr. 31, 1542-1548. 171 Nohm. L.L.. Ri~cra, J.II. and Khan, N.N. I 1'~'121Biochim. Biophy~,. Acta. (in prc~,~,). IS] Nolan. L.L. and Rivcra. J.ll. (l~H) Bi(uchem. Int. 25, 49t}-511,R. I~1 Ilolmes, A.W., Cheriatundam, E., Kalinski, A. and Chang, LM.S. 11984) Mol. Bi~chem. ParasiloL Ill. 195-21)5. 1111] Dube, D.K., Williams, G.S. and Williams, S.C. 11t1791 Bi~chim. Biophys. Acla 50,1. Itl-I6. [111 Solari. A.. Tharaun, D.. Repetto. Y.. Aldunate, J.. Morello, A. and kitvak, S. (19831 Biochem. Int. 7. 147157. [121 Abu-Elhciga, L. Spira, D.T. and Bachrach. U. 11991)) Exp. Parasilol. 71.21-26. [131 Bzik, D.J. and Fox, B.A. (1991) Mol. Biochem. Parasitol. 49, 289-290,. [141 Torri, A.F. and Englund. P.T. 119921 J. Biol. ('hem. 267. 4786-4792. [151 Matsukage, A.. Nishikav,'a. K., O~,i, T., Seto, ~. and Yamaguchi. M. 11~1871J. Biol. ('hem. 262, St~Nl-gth'~2. [10'] Wilson, S,. Ahhotts. J. and Widen. S. 119881 Biochim. Bit,phys. Acta q49. 149-157. [17] Burgers, P.M.. Bambara, R.A., Campebell. J.L.. et al. 119~1) Eur. J. Biochem. 191,617-618. [18] North. T.W. and Wyler, D. (1987) MLfl. Biochem. Parasitol. 22. 215-221. [It)] Bambara. R.A. and Jessee. C.B. I ItIS I) Bit~:him. Biophys. Acta. 10,",S. 11-24. [211] DcVrics. 2... Siam. J.G.. Frarmcn, F.F.J.. Van der Viler. P.C. and Overdulve. J.P. 119ql ) Mol. Biochem. Parasitol. 45. 223-232. [211 Talanian. R.V.. Brov,n. N.C.. McKcnna. ('.E.. Ye. T.-G.. Le~s. J.N. and Wright. G.E. (19~91 Biochemisl~' 28. 8270-8274. 122] Syvaoja. J.. Suomensaari. S.. Nishida. C.. Goldsmith. J.S.. Chui. G.S.J.. Jain, S. and Linn. S. 1lt~qtl) Prt~. Natl. Acad. Sci. USA 87. 0,0,64-0,668. [23] Byrnes. J.J.. Downe.~,. K.M.. Esserman. L. and St,. A.G. 119751 Bi~:hemistry 14. 796-79tL [24] De Clcrq. E. 1191't81. Adv. Drug Res. 17. 1-59. [25] Chang. L.M.S. and Bollum. F.J. 119811 J. Biol. Chem. 250', 494-498. [20,] Burgers. P.M.J. (198,9) Ping. Nucleic Acid.,, Res. Mol. Biol. 37. 235-2811.
FEMSLE I)4975
The D N A polymerases of Leishmania mexicana L i n d a L. N o l a n
t
a n d J o s e H. R i v e r a
Uni~'ersity of Massachusetts, &'hoolof Public Ih'alth. .4mh('r~t. MA, USA
Received 311September 1991 Revision received5 May 1092 Accepted 6 May 1092 Key words: D N A polymerase; Leishmanm mextcana: Chemotherapy
1. S U M M A R Y Two previously isolated D N A polymerases from the parasitic protozoan Leishmania mexicana were further characterized by exposure to inhibitors of mammalian D N A polymerases. D N A polymerase A, a high molecular mass enzyme, and D N A polymerase B, a /3-like D N A polymerase were compared to each other and to their mammalian counterparts regarding pH optimum, utilization of templates, and response to various inhibitors and ionic strengths. The results suggest that the D N A polymerases from L. mexicana differ from the host enzymes and may offer a target for chemotherapeutic intervention.
2. I N T R O D U C T I O N Five classes of D N A polymerase (a, /3, y, (~ and e) have been isolated from higher eukaryotic cells [1-4] and a , / 3 and y-like polymerases from t Correspondence to: Present address: L. Nolan. School of
Public Health, Morrill Science Center, Universityof Massachusetts, Amherst, MA 01IX)3,USA.
parasitic protozoa [5-14]. D N A polymcrases a, and E are nuclear enzymes associated with chromosomal replication: /3 is a low molecular mass nuclear enzyme involved in D N A repair [2,15,16], and y which has been isolated from mitochondria is believed to be responsible for mitochondrial D N A replication [2,4,17]. We have been studying D N A replication in the kinetoplast parasite Leishmania mexicana and have begun studies to characterize the major polymerase activities in these parasites for the purpose of comparing them to host polymerases, particularly a and/3. Although North and Wyler [18] reported studies of in vivo D N A replication of Leishmania parasites, this laboratory is the first to report the isolation and characterization of the leishmaniai D N A polymerases in vitro [68]. Others have described purification of a-like, /3-like [9] and y-like polymerases [14] from the parasitic protozoans Crithidia fasciculata and an a-like polymerase from Trypanosoma bmcei [10] and Trypanosoma cmzi [11]. The purpose of this study is to compare the major D N A polymerase activities (A and B) isolated from Leishmania mericana to a and /3 polymerases isolated from other sources.
72 3. MATERIALS AND METHODS
3.1. Test organism Leishmania mexicana amazonensis (Walter Reed strain 227) obtained from the Leishmania section of the Walter Reed Army Institute of Research were grown in brain-heart infusion media as previously described [6]. 3.2. Preparation of compounds Aphidicolin (Sigma, St. Louis, MO) was prepared in dimethyisulfoxide (DMSO) as a 5 mM stock and diluted with water so that the final concentration of DMSO in the assay was no more than 0.16% (v/v). Suramin, purchased from Miles Pharmaceuticals (West Haven, CT), was made into a 70 mM stock solution in 10 mM Tris, pH 7.5. Further dilutions were made with the same buffer. Butylphenyl dGTP (BuPdGTP), carbonyldiphosphonate (COMDP) and the phosphonoacetic acid derivatives BrPAA, CIPAA, FPAA and F2PAA were generous gifts from Dr. G. Wright, University of Massachusetts Medical Center (Worcester, MA), and were prepared in aqueous solution at the appropriate concentrations. All other chemicals were purchased from Sigma. 3.3. Isolation of DNA polymerases DNA polymerase A, a high molecular mass DNA polymerase sensitive to N-ethylmaleimide (NEM), was isolated from L. mexicana promastigotes as previously described [7]. A low molecular mass DNA polymerase classified as a /3-like enzyme was isolated from promastigotes as previously described [8]. This enzyme will be referred to as DNA polymerase B, to distinguish it from mammalian enzymes and to follow the designation used by Holmes et al. [9] for the Crithidia fasciculata DNA polymerases. 3.4. Drug assays The inhibitory properties of several compounds were determined by pre-incubating the enzyme and drug in the assay mix. In order to characterize the enzymes, selective inhibitors of mammalian DNA polymerases were tested against both enzymes. DNA polymerase A was assayed at
35°C as described [7]. DNA polymerase B activity was measured at 35°C as previously described [8].
4. RESULTS AND DISCUSSION
4.1. Isolation of DNA polymerases Two types of DNA polymerase activity were separated using affinity chromatography with denatured DNA cellulose. The two enzyme activities were designated as DNA polymerase A [7] and a /3-like DNA polymerase [8] according to their molecular mass, pH optimum and response to N-ethylmaleimide. The B enzyme (pol B) was less stable than the DNA polymerase A (pol A) at all stages of the purification. The use of a mixture of protease inhibitors as well as glycerol during the isolation procedures was essential for stability of the DNA polymerases. In addition, the use of a freshly prepared assay mix was critical in obtaining pol B enzyme activity. Difficulty in detecting a low molecular mass DNA polymerase in parasitic protozoans has resulted in conflicting reports from some groups regarding the presence of a low molecular mass DNA polymerase in Trypanosoma brucei [5,10]. In addition, studies on T. cruzi detected only one DNA polymerase of high molecular mass and no/I-like enzymes [11]. Proper characterization of the DNA polymerases from L. mexicana is essential in order to compare them with the host enzymes, a first step in a strategy to develop chemotherapeutic agents. To date, all enzymes isolated from parasitic protozoans have been foand to share some, but not all, of the characteristics of the mammalian enzymes [5,7-14]. 4.2. Characterization studie~ Pol A was slightly stimulated by NaCI or KCI at concentrations of less than 15 mM, but rapidly inactivated by higher concentrations of salt [7]. DNA polymerase B was slightly stimulated by 5 mM KCI only, but was more resistant to inactivation by higher concentrations of NaCI or KCI, with 35% of the activity remaining in the presence of 200 mM NaCI and 43% of the activity remaining in the presence of 200 mM KCi [8].
Table I T e m p l a t e specificity of the L. mexicana D N A polymerases A and B Template-Primer
Labelled nucleotide
Divalent cation
Activated D N A
dTTP dTTP dTTP dTTP dTTP dTTP dGTP dGTP dGTP dGTP dGTP dGTP
Mg 2 " Mn 2 ~ M g 2" Mn 2" M g 2" Mn 2 ÷ M g 2" Mn 2" Mg 2+ Mn 2 ~ M g 2" Mn 2 +
poly(rA).oligo (dT)lo I~fly(dA)" °lig°~dT);2- t~ Activated D N A poly(rC).oligo(dG) m poly(dC)- oligo(dG )t 2_ I~
c~ of activity relative to activated D N A with [ methyl- 311]dTlrP D N A POl A
D N A pol B
100 18 3 31 q9.2 27.5 1.76 11.4 (I 1.3 57.8 5.4
100 0 I).9 0.9 149 0 17 0 2 It 650 0.9
Concentrations of divalent cations were 8 m M MgCI 2 or 11.5 m M MgCI 2.1Adapted from Nolan et al. [7] and Nolan and Rivera [8].)
Table 2 IC50 values of several D N A polymerase inhibitors on the L. mt:ricana pol A and pol B [7,8] Inhibitor
DNA polyme,lse
L. mexicana
Mammalian a (~M) AraCTP Aphidicolin NEM BuPdGTP ddTTP PAA FPAA BrPAA CIPAA FzPAA COMDP Suramin Hemin
- ,, '~ < 100 0.3 R 71-7111 ¢ 20 1611 121) 300 a 300 -
/3
"),
- " R > 111 m M _ b 2 R
R < 11111 > 11111 11.115 71-7111 ¢
8
2 < 101111 > 11) R 2 2t} 70 21Xl" 411 -
~
15 > 11)
21111 ~
A (/zMI
B
R R < 1 I11X1 I1~) R 1511t) 1311 R R R 151) 8 90
> 51111 ~.M R R 5.4 7.5 R R R R R 21111 3 611
T h e IC5. values for the m a m m a l i a n D N A polymerases indicate representative data obtained from published values in the literature [2.19,21,22,25]. Enzymes found to be resistant to inhibition were m a r k e d as R. a It has been reported that, as a rule. m a m m a l i a n polymerase/3 is less sensitive than polymerase a with K i values ranging from 2 - 4 ,aM for polymerase a versus 13-32 t~M for polymerase/3 [2]. , T o 6fir knowledge there is no published data on the inhibition of polymerase/3 by B u P d G T P . However. it has been reported that p o l y m e r a s e / 3 is weakly inhibited by B u P d G T P [24]. ¢ Concentration range at which D N A polymerases a and y are reportedly inhibited by P A A [2]. d Values obtained using poly(dA).oligo~dT) I_,_ is as the template [21]. " T h e s a m e study reported 93% inhibition of D N A polymerase 8 from H e L a cells by 15 ~,M C O M D F using poly(dA)-oligo(dT) IZ%
74 Mammalian D N A polymerase a is inhibited by high ( > 100 raM) concentrations of salt, whereas D N A polymerases /3 and ,5 are stimulated by such concentrations [2]. The optimum pH of the pol A enzyme is mildly acidic to neutral at 6.7, whereas the optimum pH of the pol B enzyme is basic, at 9.0. Table I shows a comparison of the activity of the enzymes with several template-primers. The pol A had a template preference for activated D N A and used poly(dA), oligo(dT)t,_ i~ equally as well, with only 6(1% of the activity when poly(dC). oligo(dT)lz_18 was the template [7]. Pol B showed a six-fold preference for poly(dC), oligo(dT)tz_18 as the template over activated D N A [8]. The preferred template for D N A polymerases a and 13 is activated DNA, whereas the mitochondrial D N A polymerase is more active with poly(rA). oligo(dT). A notable point is the inability of pol B to utilize Mn ÷z as the divalent cation activator. In contrast, mammalian D N A polymerase /3 is capable of using both Mn 2÷ and Mg -'+ [2]. 4.3. hthibitor studies Exposure of the enzymes {o specific D N A poly~crase inhibitors showed the L. mexicana enzymes t,, be different from one another and from mammalian enzymes in their sensitivity to various compounds (Table 2). Both enzymes were resistant to aphidicglin, a mammalian D N A polymerases a, ~ and E inhibitor. The response of these enzymes to the mammalian D N A polymerase a inhibitor BuPdGTP was interesting, in the presence of 100 # M dGTP, pol B was twenty-fold more sensitive to this compound than pol A with a concentration that inhibits activity by 5(1% (IC~(,) of 5.4 p,M, whereas the pol A was inhibited with an IC50 of 100 # M . Phosphonoacetic acid (PAA) was a weak inhibitor of the pol A with only 35% inhibition at 2 raM. Pol B was resistant to PAA at concentrations of up to 2 mM. Mammalian /3 polymerase has been found to be resistant to inhibition by this compound [2]. Several PAA analogues [21] were tested against the L. mexicana D N A polymerases (Table 2). Pol B was completely resistant to inhibition by the fluoro, bromo, chloro, and difluoro analogues of PAA (FPAA, BrPAA, C!PAA, F2PAA, respec-
tively). Pol A was resistant to BrPAA, CIPAA, and F2PAA. FPAA, a monohalogenated derivative of PAA, inhibited pol A with an IC5, of 131) p,M, resulting in a greater than ten-fold increase in inhibition compared to PAA. F P A A also exhibited potent inhibition of the calf thymus D N A polymerases a and `5 (Table 2; ref. 21). COMDP, a specific inhibitor of mammalian D N A polymerase ,5 (refs. 15, 22; G. Wright, personal communication), was inhibitory to both enzymes from L. mexicana. The pol A enzyme was more sensitive to C O M D P than the pol B with ICs0s of 150 and 2011/xM, respectively (Table 2). The response of these L. mexicana enzymes to non-specific inhibitors showed the unique properties of each enzyme (Table 2). Hemin, a critical nutritional component of the leishmanial growth media [6], was found to inhibit both enzymes, inhibiting pol B with an ICs~j of 6(I # M versus an ICs~ of 91) p~M for the pol A. Heroin inhibits D N A synthesis reversibly by binding D N A polymerase and causing it to dissociate from the template [23]. Suramin, a drug used in the treatment of trypanosomiasis that has also been found to be a strong competitive inhibitor of the reverse transcriptase of a number of animal retroviruses [24], was found to be a potent inhibitor of the 1.. mexicana D N A polymerases. Suramin gave an IC5~ of 8 # M for pol A and 3 # M for pol B (Table 2). Our characterization studies have shown the L. mexicana pol A and pol B to differ from each other in molecular mass, pH optimum, template specificity, and response to salt and inhibitors. In addition, our studies have shown that pol A and pol B share similar properties such as pH optimum, molecular mass, and sensitivity to specific inhibitors such as NEM, with their mammalian counterparts. The assignment of poi A to a specific class among the eukaryotic D N A polymerases is made difficult by its utilization of template (Table 1) and by the particular response of this enzyme to inhibitors (Table 2). Although this high molecular mass enzyme shows a-like properties such as inhibition by N E M and salt, insensitivity to ddTTP, and preference for Mg 2-~ and activated DNA, it also displays characteristics that do not fit the a type. Pol A also shows
characteristics of the ~5 type, such as resistance to aphidicolin and utilization ( a l t h o u g h at low levels; Table 1) of ribonucleotide t e m p l a t e w h e n Mn-' is the divalent cation. O n the o t h e r hand, the low sensitivity to B u P d G T P and the s o m e w h a t high sensitivity to C O M D P point toward characteristics o f the D N A p o l y m e r a s e s ~ and ~ [19]. Pol B can be m o r e easily classified as a /3-like enzyme b a s e d on its low molecular mass, resistance to N E M , a n d sensitivity to d d T T P . However, pol B failed to cross-react with an anti-rec o m b i n a n t m o u s e D N A p o l y m e r a s e /3 a n t i s e r u m using e n z y m e neutralization studies [8]. Using e n z y m e neutralization studies, as well as i m m u n odiffusion and i m m u n o e l e c t r o p h o r e s i s , C h a n g and Bollum [25] s h o w e d that 7: bnu'ei D N A p o l y m c r a s e /J did not cross-react with an antis e r u m against calf t h y m u s D N A p o l y m e r a s e ft. O b s e r v a t i o n s of differences with the m a m malian p o l y m e r a s e have b e e n m a d e on the enzymes o f o t h e r p r o t o z o a n s [7-14,20,25], suggesting that D N A replication in h i g h e r e u k a r y o t e s a n d p r o t o z o a n s may differ. Such differences are being characterized in this laboratory in the s e a r c h lor potential anti-parasitic agents.
ACKNOWLEDGEMENTS W e are grateful to Drs. N a s e e m a Khan, G e o r g e Wright and Lech Dudycz for giving us advice and p o l y m e r a s e inhibitors to test. We also wish to thank Therese Lapointe for typing the m a n u s c r i p t . This w o r k was s u p p o r t e d by a contract f r o m the U.S. A r m y Medical R e s e a r c h a n d D e v e l o p m e n t C o m m a n d , D A M D 17-87-C-7146, to L.L.N.
REFERENCES [I] Sctwassi. A.I.. Plevani. P. and Bertazmni. U. 119811) Trends Biochem. Sci 5. 335-337.
[2] FrT'. M. and t.~b. L.A. (1985) Animal Cell DNA Imlymerases. ('RC Press. I~ca Raton, FL. [3] Loeb. L.A. (Itl741 In: The En~'mes. IP~yer. P.D.. Ed.). pp. 174-2119. Academic Press, Nev."York. [4] Wang, T.S.F. (1~91) Annu. Re,;. Biochem. ~11,513-552. [5] Chang, L.M.S.. Cheriatundam. E.. Mahone.v, E.M. and Ccrami. A. (1981)) Sclence 2118.510-511. [6] Nolan. L.L. I It'S71 Antimicrob. Agents C'hemotcr. 31, 1542-1548. 171 Nohm. L.L.. Ri~cra, J.II. and Khan, N.N. I 1'~'121Biochim. Biophy~,. Acta. (in prc~,~,). IS] Nolan. L.L. and Rivcra. J.ll. (l~H) Bi(uchem. Int. 25, 49t}-511,R. I~1 Ilolmes, A.W., Cheriatundam, E., Kalinski, A. and Chang, LM.S. 11984) Mol. Bi~chem. ParasiloL Ill. 195-21)5. 1111] Dube, D.K., Williams, G.S. and Williams, S.C. 11t1791 Bi~chim. Biophys. Acla 50,1. Itl-I6. [111 Solari. A.. Tharaun, D.. Repetto. Y.. Aldunate, J.. Morello, A. and kitvak, S. (19831 Biochem. Int. 7. 147157. [121 Abu-Elhciga, L. Spira, D.T. and Bachrach. U. 11991)) Exp. Parasilol. 71.21-26. [131 Bzik, D.J. and Fox, B.A. (1991) Mol. Biochem. Parasitol. 49, 289-290,. [141 Torri, A.F. and Englund. P.T. 119921 J. Biol. ('hem. 267. 4786-4792. [151 Matsukage, A.. Nishikav,'a. K., O~,i, T., Seto, ~. and Yamaguchi. M. 11~1871J. Biol. ('hem. 262, St~Nl-gth'~2. [10'] Wilson, S,. Ahhotts. J. and Widen. S. 119881 Biochim. Bit,phys. Acta q49. 149-157. [17] Burgers, P.M.. Bambara, R.A., Campebell. J.L.. et al. 119~1) Eur. J. Biochem. 191,617-618. [18] North. T.W. and Wyler, D. (1987) MLfl. Biochem. Parasitol. 22. 215-221. [It)] Bambara. R.A. and Jessee. C.B. I ItIS I) Bit~:him. Biophys. Acta. 10,",S. 11-24. [211] DcVrics. 2... Siam. J.G.. Frarmcn, F.F.J.. Van der Viler. P.C. and Overdulve. J.P. 119ql ) Mol. Biochem. Parasitol. 45. 223-232. [211 Talanian. R.V.. Brov,n. N.C.. McKcnna. ('.E.. Ye. T.-G.. Le~s. J.N. and Wright. G.E. (19~91 Biochemisl~' 28. 8270-8274. 122] Syvaoja. J.. Suomensaari. S.. Nishida. C.. Goldsmith. J.S.. Chui. G.S.J.. Jain, S. and Linn. S. 1lt~qtl) Prt~. Natl. Acad. Sci. USA 87. 0,0,64-0,668. [23] Byrnes. J.J.. Downe.~,. K.M.. Esserman. L. and St,. A.G. 119751 Bi~:hemistry 14. 796-79tL [24] De Clcrq. E. 1191't81. Adv. Drug Res. 17. 1-59. [25] Chang. L.M.S. and Bollum. F.J. 119811 J. Biol. Chem. 250', 494-498. [20,] Burgers. P.M.J. (198,9) Ping. Nucleic Acid.,, Res. Mol. Biol. 37. 235-2811.
