Trypanosoma brucei Has Two Distinct Mitochondrial DNA Polymerase β Enzymes

In higher eukaryotes, DNA polymerase (pol) β resides in the nucleus and participates primarily in DNA repair. The DNA polymerase β from the trypanosomatid Crithidia fasciculata, however, was the first mitochondrial enzyme of this type described. Upon searching the nearly completed genome data base of the related parasite Trypanosoma brucei, we discovered genes for two pol β-like proteins. One is ∼70% identical to the C. fasciculata pol β and is likely the homolog of this enzyme. The other, although ∼30% identical within the polymerase region, has unusual structural features including a short C-terminal tail and a long N-terminal extension rich in prolines, alanines, and lysines. Both proteins, when expressed recombinantly, are active as DNA polymerases and deoxyribose phosphate lyases, but their polymerase activity optima differ with respect to pH and KCl and MgCl2 concentrations. Remarkably, green fluorescent protein fusion proteins and immunofluorescence demonstrate that both are mitochondrial, but their locations with respect to the mitochondrial DNA (kinetoplast DNA network) in this organism are strikingly different. In higher eukaryotes, DNA polymerase (pol) β resides in the nucleus and participates primarily in DNA repair. The DNA polymerase β from the trypanosomatid Crithidia fasciculata, however, was the first mitochondrial enzyme of this type described. Upon searching the nearly completed genome data base of the related parasite Trypanosoma brucei, we discovered genes for two pol β-like proteins. One is ∼70% identical to the C. fasciculata pol β and is likely the homolog of this enzyme. The other, although ∼30% identical within the polymerase region, has unusual structural features including a short C-terminal tail and a long N-terminal extension rich in prolines, alanines, and lysines. Both proteins, when expressed recombinantly, are active as DNA polymerases and deoxyribose phosphate lyases, but their polymerase activity optima differ with respect to pH and KCl and MgCl2 concentrations. Remarkably, green fluorescent protein fusion proteins and immunofluorescence demonstrate that both are mitochondrial, but their locations with respect to the mitochondrial DNA (kinetoplast DNA network) in this organism are strikingly different. Trypanosoma brucei belongs to a family of parasitic protozoa that are responsible for a growing human health problem. T. brucei is the causative agent of African sleeping sickness, a disease that threatens millions of people in sub-Saharan Africa, and related species are responsible for diseases such as Chagas disease and Leishmaniasis. Trypanosomes are among the earliest diverging eukaryotes that contain a mitochondrion (1.Cavalier-Smith T. Trends Genet. 1997; 13: 6-9Abstract Full Text PDF PubMed Scopus (38) Google Scholar, 2.Sogin M.L. Curr. Opin. Genet. Dev. 1991; 1: 457-463Crossref PubMed Scopus (396) Google Scholar), and perhaps because of this niche in evolution they exhibit some unique biological hallmarks. A prominent example is the fascinating structure and organization of their mitochondrial genome, known as kinetoplast DNA (kDNA). 1The abbreviations used are: kDNAkinetoplast DNApolpolymerasedRP5′-deoxyribose phosphatedTMPdeoxythymidine 5′-monophosphatePMSFphenylmethylsulfonyl fluorideGFPgreen fluorescent protein.1The abbreviations used are: kDNAkinetoplast DNApolpolymerasedRP5′-deoxyribose phosphatedTMPdeoxythymidine 5′-monophosphatePMSFphenylmethylsulfonyl fluorideGFPgreen fluorescent protein. The kDNA contains several thousand DNA minicircles catenated together into a large network resembling medieval chain mail in its topology. In vivo this network is tightly condensed into a disk-shaped structure that associates with the basal body of the single flagellum of the parasite (see Ref. 3.Shapiro T.A. Englund P.T. Annu. Rev. Microbiol. 1995; 49: 117-143Crossref PubMed Scopus (284) Google Scholar for a review on kDNA). kinetoplast DNA polymerase 5′-deoxyribose phosphate deoxythymidine 5′-monophosphate phenylmethylsulfonyl fluoride green fluorescent protein. kinetoplast DNA polymerase 5′-deoxyribose phosphate deoxythymidine 5′-monophosphate phenylmethylsulfonyl fluoride green fluorescent protein. With the topological constraints imposed by the unusual network organization of this mitochondrial DNA, it is difficult to imagine a priori how this genome might be replicated in an orderly fashion. Several proteins involved in this process have been identified, and they have distinct localizations with respect to the kDNA disk. This organization suggests the events of replication are spatially regulated, and knowledge of the functions and locations of these proteins has led to a fairly detailed model for kDNA replication (for recent reviews see Refs. 4.Klingbeil M.M. Drew M.E. Liu Y. Morris J.C. Motyka S.A. Saxowsky T.T. Wang Z. Englund P.T. Protist. 2001; 152: 255-262Crossref PubMed Scopus (43) Google Scholar and 5.Morris J.C. Drew M.E. Klingbeil M.M. Motyka S.A. Saxowsky T.T. Wang Z. Englund P.T. Int. J. Parasitol. 2001; 31: 453-458Crossref PubMed Scopus (52) Google Scholar). Briefly, minicircles are released from the network, presumably by a topoisomerase, prior to their replication. Release is vectorial into the kinetoflagellar zone, the space between the kDNA disk and the mitochondrial membrane nearest the flagellar basal body (6.Drew M.E. Englund P.T. J. Cell Biol. 2001; 153: 735-744Crossref PubMed Scopus (58) Google Scholar). In the kinetoflagellar zone, they encounter the origin binding protein UMSBP (7.Abu-Elneel K. Robinson D.R. Drew M.E. Englund P.T. Shlomai J. J. Cell Biol. 2001; 153: 725-734Crossref PubMed Scopus (57) Google Scholar), a DNA primase (8.Li C. Englund P.T. J. Biol. Chem. 1997; 272: 20787-20792Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), two Pol I-like DNA polymerases (Pol IB and Pol IC) (9.Klingbeil M.M. Motyka S.A. Englund P.T. Mol. Cell. 2002; 10: 175-186Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), and likely other proteins that promote replication initiation. Replication via a theta structure intermediate is unidirectional and as such results in sister progeny containing either a single gap in the continuously synthesized strand or multiple gaps between Okazaki fragments on the discontinuously synthesized lagging strand. It is not known whether minicircle replication is completed in the kinetoflagellar zone, but eventually newly replicated minicircles migrate to two discrete sites flanking the kDNA disk known as the antipodal sites. Here they encounter SSE1 (10.Engel M.L. Ray D.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8455-8460Crossref PubMed Scopus (48) Google Scholar), an enzyme with RNase H activity that can remove RNA primers (11.Hines J.C. Engel M.L. Zhao H. Ray D.S. Mol. Biochem. Parasitol. 2001; 115: 63-67Crossref PubMed Scopus (14) Google Scholar), and DNA polymerase β (12.Ferguson M. Torri A.F. Ward D.C. Englund P.T. Cell. 1992; 70: 621-629Abstract Full Text PDF PubMed Scopus (105) Google Scholar), which presumably fills in most of the gaps between Okazaki fragments. Once this processing takes place, the newly replicated minicircles are reattached to the periphery of the kDNA network by a topoisomerase II (13.Wang Z. Englund P.T. EMBO J. 2001; 20: 4674-4683Crossref PubMed Scopus (124) Google Scholar) that also localizes to the antipodal sites (14.Melendy T. Sheline C. Ray D.S. Cell. 1988; 55: 1083-1088Abstract Full Text PDF PubMed Scopus (127) Google Scholar). Interestingly, one or two gaps are retained on the newly replicated minicircles after they have been reattached to the network (15.Ryan K.A. Englund P.T. Mol. Cell. Biol. 1989; 9: 3212-3217Crossref PubMed Scopus (27) Google Scholar, 16.Ryan K.A. Englund P.T. J. Biol. Chem. 1989; 264: 823-830Abstract Full Text PDF PubMed Google Scholar). The residual gaps are thought to represent a counting mechanism that distinguishes molecules that have completed replication from those that have not yet begun the process (and are still covalently closed). Once the entire network has been replicated, the remaining gaps are filled, and the network is divided for segregation of the progeny into daughter cells. Although much of what we know of kDNA replication has come from biochemical studies of purified proteins from the organism Crithidia fasciculata, the advent of the T. brucei genome project has facilitated a genomics approach to identify other replication proteins. In a search for the mitochondrial pol β homolog from T. brucei, we also anticipated finding a conventional nuclear enzyme as well. We report here the discovery of two pol β proteins in T. brucei, but remarkably both target to the mitochondrion. These two proteins have different intramitochondrial localizations with respect to the kDNA disk and prefer strikingly different buffer conditions for optimal polymerase activity. These differences suggest that the two pol βs have distinct nonredundant roles. The discovery of this second mitochondrial pol β brings the tally of trypanosome mitochondrial DNA polymerases to the astounding number of six, including the previously described pol β (17.Torri A.F. Englund P.T. J. Biol. Chem. 1995; 270: 3495-3497Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) and four pol I-like polymerases (9.Klingbeil M.M. Motyka S.A. Englund P.T. Mol. Cell. 2002; 10: 175-186Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Identification and Cloning of Full-length Genes—A TBLASTN search of the T. brucei genome data bases (www.tigr.org/tdb/mdb/tbdb and (www.sanger.ac.uk/Projects/T_brucei/) using the C. fasciculata pol β protein sequence as the query pulled out two distinct sets of sequences. These sequences were used to design primers, and the full-length coding sequences for both genes were acquired from PCR amplification of genomic DNA from the T. brucei cell line 29-13. The primers used are indicated in subsequent sections. All of the PCRs utilized 30 cycles of 94 °C melting (30 s), 55 °C annealing (30 s), and 72 °C extension (3 min). The accession numbers are AY354517 for pol β and AY354516 for pol β-PAK, the second pol β. Purification of Recombinant Proteins—The coding sequence for T. brucei pol β lacking the putative mitochondrial localization signal (first 11 amino acids, based on homology to the C. fasciculata pol β) was amplified using the primers B-Nde(F) (5′-CATATGCTCAGACGAACCTTCTTC) and B-Bam(R) (5′-GGATCCCTATGGGTCCCGATTCTC) and cloned into the pET14b expression vector between the NdeI and BamHI restriction sites allowing for expression of an N-terminal His6-tagged protein. After electroporation into Escherichia coli BL21(DE3), expression of the tagged protein in a 2-liter culture was induced for 3.5 h at 37 °C with 1 mm isopropylthio-β-d-galactoside. The cells were harvested by centrifugation, resuspended in 30 ml of buffer A (50 mm Hepes, pH 7.6, 5 mm EDTA, 50 mm KCl, 1 mm PMSF) supplemented with 1 mg/ml lysozyme (Sigma), and incubated on ice for 30 min. The cells were lysed by sonication (six 10-s bursts at full power using a Branson sonicator). The lysates were centrifuged (30,000 × g, 15 min, 4 °C), and the resulting supernatants were applied to a 5-ml DEAE-cellulose column that had been equilibrated in buffer A. The column was with 15 ml of buffer and the and were and applied to a 5-ml column also equilibrated in buffer A. The column was with ml of buffer and protein was with a to containing pol β were buffer (50 mm Hepes, pH 7.6, mm KCl, mm 1 mm and applied to a column equilibrated with buffer The column was with ml of buffer and protein was with 5 ml of buffer containing mm of the the pol β protein was by using a the C. fasciculata pol β T.T. Y. Englund P.T. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) which with the T. brucei protein. The purified protein was buffer containing 50 mm Hepes, pH 7.6, 50 mm KCl, 1 mm EDTA, and 1 mm After was to and the was at This of protein as using a the purified protein by and with The entire coding sequence for T. brucei pol was using the primers and and cloned into the of the pET14b This was into and protein expression was induced in of culture by the of 1 mm for h at The cells were harvested and and the was a column as described The and were applied to a 5-ml column equilibrated in buffer A. The column was with ml of buffer and protein was with 15 ml of buffer A containing or mm The mm KCl which pol β-PAK, was applied to a column that had been equilibrated with buffer (50 mm Hepes, pH 7.6, mm KCl, mm and 1 mm The column was with ml of buffer and protein was with 5 ml of buffer supplemented with mm of the the pol protein was by using a that the The purified protein was buffer lacking and to in a was to and the resulting protein was at This of protein. The protein by and is in Recombinant C. fasciculata pol β was as described previously C. H. J. Cell Sci. PubMed Google Scholar). that a in the sequence of the C. fasciculata pol β has been in number to pol β were in protein. pol β-PAK, were in Although the was with the protein of the subsequent were with containing the full-length protein. All of the were at The were after four a of and cells were harvested by at × for 5 min. cells were in The cell were resuspended in and 5 × cell were and the proteins were to a membrane by The were in mm pH mm and supplemented with for 1 h and with for pol β and for pol into was for an 1 The were for in and incubated with for pol β and for pol β-PAK, both to from for 1 were into containing The were for with and the were by of base and were described previously C. H. J. Cell Sci. PubMed Google Scholar). The was and the enzyme were 1 was on and the was by conditions were previously described C. H. J. Cell Sci. PubMed Google Scholar), that the was of a In optimal buffer one was from the buffer conditions (50 mm pH 5 mm mg/ml in the pH was of the were of Cell sequences for were amplified by PCR using the primers and for pol β and and for pol The PCR were cloned into the vector A Scholar) of between the and restriction enzyme sites to the C-terminal fusion protein. This vector of the fusion protein with a The vector was with to for into the into and of cells were described previously (9.Klingbeil M.M. Motyka S.A. Englund P.T. Mol. Cell. 2002; 10: 175-186Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). were described previously (13.Wang Z. Englund P.T. EMBO J. 2001; 20: 4674-4683Crossref PubMed Scopus (124) Google Scholar) that cells were in and was (Pol that had been on to T. J. C. J. Cell Biol. 1997; Google Scholar) was into containing and applied to for min. The were incubated with to for min. was as previously described (6.Drew M.E. Englund P.T. J. Cell Biol. 2001; 153: 735-744Crossref PubMed Scopus (58) Google Scholar) using an The were acquired with a using T. brucei pol β-like search of the T. brucei genome data base two on that pol β proteins. A of the proteins by the two pol β genes is with that of the C. fasciculata protein in The of the two a protein that is ∼70% identical to the C. fasciculata pol including a putative mitochondrial signal at the The two proteins are likely and we have the trypanosome protein as T. brucei pol β. The other a much protein of the polymerase this protein ∼30% sequence with both the C. fasciculata and T. brucei pol β proteins. Interestingly, this protein has a C-terminal tail as as a N-terminal extension rich in prolines, alanines, and both of which are in the other proteins. of the we have this protein as T. brucei pol Although we this second pol β to be it also contains a putative mitochondrial signal at its based on sequence previously J.C. Ray D.S. Mol. Biochem. Parasitol. PubMed Scopus Google Scholar). The sequence of the has unusual It for of the and of its sequence is of the amino and pH it is to a of this are in A search using this sequence out homology from but its amino is of two proteins cloned from C. fasciculata, protein 1 A.F. Englund P.T. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar) and DNA J. C. and T. Interestingly, both of these proteins are mitochondrial and with the Both and pol β and pol whether expression of either pol β or pol was on the of the we used to cell lysates from both and The proteins were to a membrane and using containing the proteins The β a of in both and in with the of this enzyme The a of in lysates from both This protein with for its is likely by the large of the and the of be Recombinant pol β and pol as DNA of the sequences of T. brucei pol β and pol that amino for DNA polymerase activity are in both not whether they are we proteins using buffer conditions previously for the C. fasciculata mitochondrial pol β Englund P.T. J. Cell Biol. PubMed Scopus Google Scholar). enzyme indicated that the T. brucei pol β and C. fasciculata pol β have of polymerase activity In T. brucei pol as much activity these whether the polymerase activity of pol was an of the protein or a of the buffer conditions we the of KCl and MgCl2 or the pH of the the T. brucei pol β and the C. fasciculata pol β The of both were at KCl and MgCl2 and their activity at pH T. brucei pol β-PAK, however, the optimal buffer conditions from the pol β proteins. The activity of pol was by higher of KCl and it was much of in pH the The buffer the polymerase activity most was KCl In pol the activity mm it was to of the activity by the C. fasciculata pol β (50 mm of These data that pol is not active as a DNA polymerase pol β. Recombinant pol β and pol the amino for activity are also we to whether both pol β and pol are active in this We incubated the proteins with a for or 5 and the with (see in Ref. C. H. J. Cell Sci. PubMed Google Scholar for the mechanism of the were by and the and were by both pol β and pol β-PAK, at when enzyme and were the intermediate and the enzyme was incubated with for 5 prior to much of the intermediate had been to the intermediate 4 and or the (see This was to that for the C. fasciculata pol β used as a 1 and We also the the of the by of the proteins, of this was to the was at and of when the were to for 5 prior to however, and this was and both pol β and pol can as pol β and pol Both the protein sequences of both T. brucei pol β-like genes putative mitochondrial localization J.C. Ray D.S. Mol. Biochem. Parasitol. PubMed Scopus Google Scholar), we to the of these proteins. We cloned the coding sequence of into the allowing to pol β and pol as C-terminal of the We the of the fusion proteins by of cells. of T. brucei cells the pol fusion protein of cells in which the at two flanking the kDNA disk two All cells this of had one nucleus and one kDNA and were likely in the T. brucei pol β localizes to the antipodal sites to its homolog in C. fasciculata (12.Ferguson M. Torri A.F. Ward D.C. Englund P.T. Cell. 1992; 70: 621-629Abstract Full Text PDF PubMed Scopus (105) Google Scholar). In cells with a signal to the kDNA was of pol was in cells in which the nucleus had also not its expression is by the the pol fusion protein is to the and at we some of into the entire mitochondrial not In cells in which the two antipodal sites were not this was still The antipodal localization in a of cells its C. fasciculata homolog T.T. Y. Englund P.T. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), in this is cell although we have to this protein we were to immunofluorescence data with a of and we T. brucei cells the pol fusion we were to that this protein not in the antipodal sites but to the kDNA disk using full-length pol this localization In higher eukaryotes it is that pol β is a nuclear enzyme involved in DNA repair. The pol β from the trypanosomatid parasite C. fasciculata was the first example of a mitochondrial enzyme of this type (12.Ferguson M. Torri A.F. Ward D.C. Englund P.T. Cell. 1992; 70: 621-629Abstract Full Text PDF PubMed Scopus (105) Google Scholar, A.F. Englund P.T. J. Biol. Chem. 1995; 270: 3495-3497Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, A.F. T.A. Englund P.T. J. Biol. Chem. Full Text PDF PubMed Google Scholar). We demonstrate here that the related parasite T. brucei has two genes pol β-like proteins, and both to the mitochondrion. Interestingly, data base have yet to a pol β that might represent the nuclear homolog in this the parasite Y. PubMed Scopus (44) Google Scholar), T. brucei a nuclear pol β and has other to its nuclear the pol β homolog from also a trypanosomatid has been to to the nucleus S. T. M. 2001; PubMed Scopus (27) Google Scholar). This localization that the protein is identical to the C. fasciculata mitochondrial pol β the and an N-terminal mitochondrial localization Although pol β and pol ∼30% sequence including the active for both the DNA polymerase and are differences between these two One is the long N-terminal extension rich in the amino and for which pol is This is and contains of The in this protein to the kDNA disk or it in this this a in the of this protein in vivo (see of the of between T. brucei and related we were to identify pol of the pol β genes in both the and Trypanosoma Interestingly, although the coding sequences of the second N-terminal in both the the sequence of the is the short C-terminal tail is also in these We to identify a pol β that might represent the nuclear enzyme in genome of these as well. T. brucei pol β and pol have different buffer in in polymerase Although pol β of KCl and and its activity at pH the polymerase activity of pol is by higher KCl and MgCl2 and is to The by pol in the enzyme by an or the of the enzyme. The of pol β-PAK, as as its for the that this enzyme to a second T. brucei mitochondrial polymerase activity previously J. J. J. Microbiol. PubMed Scopus Google Scholar). Although are some differences (for in optimal MgCl2 it be that these were on mitochondrial The second active have pol β-PAK, one or of the pol I-like polymerases (9.Klingbeil M.M. Motyka S.A. Englund P.T. Mol. Cell. 2002; 10: 175-186Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), or other proteins that might polymerase activity. although both pol β and pol in the they have different localizations with respect to the kDNA disk. These data suggest that these two pol β proteins have distinct and nonredundant in kDNA replication or We the model to the for two pol βs to of the minicircles in the network are covalently are released from the network and replicated, to one daughter with a single gap and one with multiple gaps Although the molecules are of network with or Ray D.S. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Englund P.T. J. Biol. Chem. Full Text PDF PubMed Google Scholar), the some processing in the antipodal sites prior to their to the kDNA In this processing pol β is thought to of the gaps between Okazaki one or two on the molecules that are reattached K.A. Englund P.T. J. Biol. Chem. 1989; 264: 823-830Abstract Full Text PDF PubMed Google Scholar). replication the of covalently minicircles and the of molecules the network of this pol is to these remaining Once the gaps are the network in two and the progeny are to daughter cells. gap not minicircle replication is be a mechanism to pol activity the We are this model by the in vivo functions of these two using an RNA In higher eukaryotes, one DNA polymerase has been to be involved in mitochondrial DNA replication and PubMed Scopus Google Scholar). the discovery of a mitochondrial pol β in a trypanosomatid organism was and report of a second mitochondrial pol β enzyme is although the pol is lacking in these a family of four pol I-like DNA polymerases into their mitochondrion (9.Klingbeil M.M. Motyka S.A. Englund P.T. Mol. Cell. 2002; 10: 175-186Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). the tally of mitochondrial DNA polymerases in at six, an The unusual and structure of the as as the of its to the to a to that this mitochondrial genome is We for