Identification and Characterization of Two DNA Polymerase Activities Present in Trypanosoma brucei Mitochondria

J. Euk. Mtcrobiul.. 4514).1998 pp. 404-410 0 199X by the Society of Protoroologirts

Identification and Characterization of Two DNA Polymerase Activities Present in Trypanosoma brucei Mitochondria JAHELY FUENMAYOR, JUN ZHANG, WILLIAM RUYECHAN AND NOREEN WILLIAMS' Deparrment of Microbiologx and Markey Cetiter,for Microbial Pathogenesis. State U n i v r r s i ~of New York, Buffalo, New York 14214, USA

ABSTRACT. We have identified and partially purified two DNA polymerase activities from purified Trjpanosorna brucei mitochondrial extracts. The DNA polymerase activity eluted from the single-stranded DNA agarose column at 0.15 M KCI (polymerase M1) was significantly inhibited by salt concentrations greater than 100 mM. utilized Mg" in preference to Mn2+as a cofactor on deoxytibonucleotide templates with deoxyribose primers, and in the presence of Mn" favored a tibonucleotide template with a deoxyribose primer. A 44 kDa peptide in this fraction crossreacted with antisera against the Crirhidiafasriculata P-like mitochondrial polymerase. In activity gels the catalytic peptide migrated at an apparent molecular weight of 35 kDa. The DNA polymerase activity present in the 0.3 M KCl DNA agarose fraction (polymerase M21 exhibited optimum activity at 120-180 mM KCI, used both Mg2+and Mn2+ as cofactors, and used deoxyribonucleotide templates primed with either deoxyribose or ribose oligomers. Activity gel assays indicate that the native catalytic peptide(s) is -80 kDa in size. The two polymerases showed different sensitivities to several inhibitors: polymerase M1 shows similarities to the Crithidia.fasciculatu P-like mitochondrial polymerase while polymerase M2 is a novel, salt-activated enzyme of higher molecular weight. Supplementary key words. Deoxyribonucleotide. enzymes, oligomers, polymerase inhibitors, ribonucleotide, templates.

W E classes of DNA polymerases, designated a,P, y, 6 and E, have been isolated from a variety of eukaryotic sources [2, 7, 191. DNA polymerases a, 6, and E are involved in replication of chromosomal DNA while polymerase @ is involved in repair of chromosomal DNA. All of these polymerases are localized in the nucleus. The y polymerases are involved in replication of mitochondrial DNA. Each group of polymerases is identified by several distinctive features including size, subunit structure, cellular location, divalent metal ion requirements, processivity, fidelity, associated enzymatic activities, and sensitivity to a variety of inhibitors [ 7 ] . Several laboratories have reported the identification and characterization of DNA polymerases from trypanosomatids. These include high molecular weight a type polymerases from Crithidia fasciculata, Trypanosoma brucei, Trypanosoma cruzi, and Leishmania sp. and low molecular weight P-like polymerases from these same organisms [ 3 , 5, 8-9, 15-16, 201. However, the properties of these polymerases frequently differ significantly from their presumed eukaryotic homologues in terms of monovalent and divalent cation requirements and sensitivity to inhibitors. They also frequently fail to show crossreactivity with polyclonal antisera raised against polymerases from higher eukaryotes. In addition to the enzymes listed above, a novel high molecular weight polymerase has been reported from Leishmania mexicana [ 131. Recently, P-like polymerases from C. fasciculata and T. cruzi have been purified to homogeneity [23-25, 271. Surprisingly, p-like DNA polymcrase from C. fuscicitlutu has been shown to be localized in the lunetoplast [6, 231, the unique mitochondrial DNA network possessed by trypanosomatids. This enzyme has very limited processivity and poor fidelity and it is believed to play a role in the repair of mitochondrial DNA replication intermediates [ 2 5 ] .Thus far, neither a nuclear P-like polymerase nor a mitochondrial P-like polymerase have been unambiguously identified in T. brucei. In the present work we report the identification of two polymerase activities isolated from the mitochondria of the procyclic form of T. brucei, one resembling in part the C. fasciculata @-likepolymerase and the second being a novel, salt-stimulated activity of higher molecular weight.

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were cultured at 27" C in Cunningham's medium [4] with 25 mM HEPES and 5% fetal bovine serum (Sigma) and 30 pg/ml

gentamicin (GIBCOBRL). The trypanosomes were collected by centrifugation once they attained logarithmic growth. Protein purification. Purification of mitochondria from 12 liters of T. brucei cultures at a density of 8 X lo6 cells per ml was performed as described by Rohrer et al. [14], and Williams and Frank [28] using Percoll density gradient centrifugation. The final mitochondrial pellet was resuspended at 4" C in mitochondrial lysis buffer (200 mM Tris HC1, pH 8.0, 20 mM EDTA, 10 mM DTT, 40% v/v glycerol, 10% v/v Nonidet-40, 4 mM phenylmethylsulfonylfluoride(PMSF), 20 pg/ml leupeptin). This lysate was dialyzed overnight against Buffer A (0.15 M KCl, 50 mM Tris HCl, 5 mM EDTA, 0.5 mM DTT, 0.2% Nonidet P-40) in the presence of protease inhibitors. Cell debris and precipitates were removed by sedimentation at 80,000 g for 30 min at 4" C . The supernatant was applied to a single-stranded DNA agarose column (5 ml bed volume, GIBCOBRL), which had been equilibrated in buffer A. The column was washed with 100 ml of Buffer A and then eluted with a step gradient consisting of 0.3 M, 0.5 M, 1.0 M, and 2.0 M KCl in Buffer A. Protein elution was monitored by absorbance at 280 nm. The total peak volume (10-15 ml) of absorbance at each salt step was collected and then concentrated to 0.15-0.40 ml using Centricon- 10 microconcentrators (Amicon). The 0.15 M KCl flow-through of the DNA agarose column was diluted 3-fold with Buffer A containing no KC1 and reapplied to the single-stranded DNA column, which had been equilibrated with Buffer A containing 50 mM KCl. The column was then eluted with 0.15 M and 2.0 M KC1, and the fractions showing absorbance at 280 nm were concentrated as described above. Polymerase assays. Activated DNA was used as a substrate (unless otherwise indicated), and was prepared according to the method of Schlabach et al. [18]. Oligo dTlpoly dA, oligo dT/ poly A, poly Alpoly dT, and poly dT were purchased from Pharmacia and stored according to the manufacturer's instructions. Oligo rA,,.,, was synthesized at the BIC FacilityAJSUHS, Bethesda, MD. Standard polymerase activity assays (SO p1 volume) contained 15 pg activated DNA, 50 mM HEPES pH 8.0, 8 mM MgC12, 1 mM dATP, 1 mM dGTP, 1 mh4 dCTP, 0.2 mM MATERIALS AND METHODS 'H-dTTP/dTTP (836 dpdpmole), 2 mM P-mercaptoethanol, Cell growth. T?ypanosoma brucei briccei strain TREU 667 and 5-10 ~1 of the polymerase fraction. As required in specific [ 101 is maintained routinely in the laboratory. Procyclic forms experiments, 0.7.5 M KCl was added to the initial reaction mixes. The reactions proceeded for 1 h at 37" C. They were ter' To whom correspondence should be addressed. Telephone: 716-829- minated by transferring the reaction mixtures to an ice bath, 2279; Fax: 7 16-829-2376: Emai!: NW 1 @acsu.buffalo.edu immediately spotting them onto Whatman GF/C filters that

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were placed in cold 5% TCA/10 mM sodium pyrophosphate and stirred for 10 min. The filters were placed in cold 1 M HCl for 10 min, then 95% ethanol for 5 min, and finally dried under an infrared heat lamp for 20 min [ 171. The incorporation of 3HdTMP was determined by liquid scintillation counting of the filters. Assays to determine optimal concentrations of monovalent and divalent cations were performed by varying the concentration of that component as described in Results. One unit of enzyme activity was defined as the amount of polymerase required to incorporate 1 pmole of TMP per hour into acid insoluble material at 37" C when activated DNA is used as a substrate. All enzyme assays were linear over a one-hour time period. Inhibitor assays. N-ethylmaleimide (NEM) inhibition was examined by preincubation of sample fractions for 1 h at 4" C prior to assays. The concentration of NEM refers to the concentration during preincubation with the polymerase. Other inhibitors were added to the reaction mixture immediately prior to addition of the polymerase samples and concentrations refer to their final concentrations in the assay. In-gel activity assay. The in-gel polymerase activity assays (IGPA) were performed as described by Blank et al. [l]. Briefly, electrophoresis was performed using a 7.5% acrylamide/0.2% bisacrylamide separating gel containing 50 pg/ml fibrinogen and 2.0 A,, units/ml of activated DNA. Protein samples containing polymerase activity were adjusted to 65 mM Tris HCl, pH 7.5, 1% SDS, 1% P-mercaptoethanol, 2 mM EDTA, and 10% glycerol. Then they were incubated for 3 min at 37" C and immediately applied to the gel [21]. SDS was removed from the gel following electrophoresis by four, sequential 15 min washes with gentle agitation in Buffer B (0.05 M Tris HCl, pH 7.5, 0.01 M P-mercaptoethanol, 0.001 M EDTA). Following removal of detergent, the gels were renatured at 37" C for 3 h, then at 4" C for 19-25 h in 0.05 M Tris-HC1, pH 7.5, 0.005 M MgCl,, 0.07 M KCI, 16% glycerol, 40 pg/ml BSA, 0.005 M pmercaptoethanol, 0.01 mM EDTA, 100 pM dCTP They were then transferred into the same renaturation buffer including 14 pM each dATP, dGTP, dTTP, 1 p,M dCTP, and 0.8 pCi/ml 32PdCTP and incubated at 37" C for 19-21 h. Gels were then washed for 1 h with gentle agitation in 4 changes of 5% TCA10 mM sodium pyrophosphate, then transferred to 4" C for 4448 h with one additional change of solution at approximately 24 h. Gels were dried and autoradiography was performed. Western analysis. Western blot analysis was performed as described [26] using primary antibodies raised against the purified C. fasciculata b-like polymerase [23] (gift of Dr. l? Englund) and antibodies raised against the T. brucei F, component of the mitochondrial ATP synthase [29] and the T. brucei nuclear p34 protein [30, 311. Antibody reactivity was visualized using horseradish conjugated secondary antibody with chloronapthol as substrate.

RESULTS Identification of two DNA polymerase activities from the mitochondria of procyclic Trypanosoma brucei. Mitochondrial extracts obtained from procyclic T. brucei were prepared as described in Materials and Methods using Percoll gradients. This results in a preparation that is free from contamination with components from other organelles as determined by studies using marker enzymes and antibodies [14, N. W., unpubl. data]. Torri and Englund 1231 used this method as a check to be certain that the @-likepolymerase they had purified from a higher yielding but cruder mitochondrial preparation was in fact present in C. fasciculata mitochondria rather than a nuclear contaminant. A Western blot of a crude whole cell extract and the mitochondrial fraction used in this purification (Fig. 1) was

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Fig. 1. Western blot analysis of crude, whole cell extract and the purified mitochondrial fraction from Trypano.snrna brucei. Lanes 1 and 2 contain 10 pg and 35 pg, respectively, of protein from whole cell extract and lanes 3 and 3 contain 7 pg and 24 pg, respectively, of protein from the mitochondria1 fraction. A . Blot probed with antibody raised against the F,, component of the mitochondrial ATP synthase. B. An identical blot probed with antibody raised against the nuclear p34 RNA-binding protein. This antibody also recognizes the closely related p37 protein. The migration of the p37 and p34 proteins is indicated by the upper and lower arrows, respectively.

probed with antibodies against the T. brucei F, component of the mitochondrial ATP synthase [29] and with antibodies which recognize the nuclear p34 and p37 RNA-binding proteins recently isolated in this laboratory [30, 311. Reactivity to the 34 kDa F, subunit of the mitochondrial ATP synthase is present in both fractions whereas the antibodies against the nuclear proteins only show reactivity in the crude extract (Fig. 1). These results confirm the lack of nuclear contamination in the mitochondrial fraction. Following dialysis against buffer containing 0.15 M KCI, the extract was adsorbed to a single-stranded DNA agarose column. Eukaryotic DNA polymerases that bind to DNA under these salt conditions fall into the p and y categories [ 2 , 71. We therefore reasoned that our initial chromatographic conditions would enrich for mitochondrial polymerases. The column was eluted with salt steps of 0.3 M, 0.5 M, 1.0 M and 2.0 M. The protein containing fractions from these salt steps were analyzed. DNA polymerase activity was detected in the 0.15 M KC1 flow-through and in the 0.3 M KCl eluate (Table 1). No significant levels of DNA polymerase activity were detected in the fractions eluted at the higher salt concentrations. The overall purification of the enzyme in the 0.3 M fraction achieved by this procedure was 177-fold based on specific activity. Repeated chromatography of the flow-through fraction on the single-stranded DNA column at the same salt concentration did not result in retention of additional polymerase activity on the column. This indicated that the activity was not due to overloading of the column. The flow-through fraction was then diluted with Buffer A containing no KCI, and adsorbed to the DNA agarose column that had been equilibrated in Buffer A containing 0.05 M KCl. The column was eluted

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Table 1. Purification of the mitochondria1 polymerase activities of Tqpunosoma hrucei. Volume Fraction Crude extract Mitochondria1 fraction

(ml)

Protein (me)

49.0

773

Total activity IU1

27,800

Specific activity fLJ/mnl

36.0

35.0

96.8

23.600

67.0 0.40 0.37 0.15 0.62

94.1 0.12 0.02 0.04