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Twinkle, the Mitochondrial Replicative DNA Helicase, Is Widespread in the Eukaryotic Radiation and May Also Be the Mitochondrial DNA Primase in Most Eukaryotes

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Abstract

Recently, the human protein responsible for replicative mtDNA helicase activity was identified and designated Twinkle. Twinkle has been implicated in autosomal dominant progressive external ophthalmoplegia (adPEO), a mitochondrial disorder characterized by mtDNA deletions. The Twinkle protein appears to have evolved from an ancestor shared with the bifunctional primase-helicase found in the T-odd bacteriophages. However, the question has been raised as to whether human Twinkle possesses primase activity, due to amino acid sequence divergence and absence of a zinc-finger motif thought to play an integral role in DNA binding. To date, a primase protein participating in mtDNA replication has not been identified in any eukaryote. Here we investigate the wider phylogenetic distribution of Twinkle by surveying and analyzing data from ongoing EST and genome sequencing projects. We identify Twinkle homologues in representatives from five of six major eukaryotic assemblages (“supergroups”) and present the sequence of the complete Twinkle gene from two members of Amoebozoa, a supergroup of amoeboid protists at the base of the opisthokont (fungal/metazoan) radiation. Notably, we identify conserved primase motifs including the zinc finger in all Twinkle sequences outside of Metazoa. Accordingly, we propose that Twinkle likely serves as the primase as well as the helicase for mtDNA replication in most eukaryotes whose genome encodes it, with the exception of Metazoa.

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References

  • Baldauf SL, Roger AJ, Wenk–Siefert I, Doolittle WF (2000) A kingdom-level phylogeny of eukaryotes based on combined protein data. Science 290:972–977

    Article  CAS  PubMed  Google Scholar 

  • Biswas TK, Getz GS (2002) Import of yeast mitochondrial transcription factor (Mtf1p) via a nonconventional pathway. J Biol Chem 2002 277:45704–45714

    Article  CAS  Google Scholar 

  • Cavalier-Smith T (2004) Only six kingdoms of life. Proc R Soc Lond B 271:1251–1262

    Article  CAS  Google Scholar 

  • Cermakian N, Ikeda TM, Cedergren R, Gray MW (1996) Sequences homologous to yeast mitochondrial and bacteriophage T3 and T7 RNA polymerases are widespread throughout the eukaryotic lineage. Nucleic Acids Res 24:648–654

    Article  CAS  PubMed  Google Scholar 

  • Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson JD (2003) Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res 31:3497–3500

    Article  CAS  PubMed  Google Scholar 

  • Clement SL, Koslowsky DJ (2001) Unusual organization of a developmentally regulated mitochondrial RNA polymerase (TBMTRNAP) gene in Trypanosoma brucei. Gene 272:209–218

    Article  CAS  PubMed  Google Scholar 

  • Desai SD, Pasupathy K, Chetty KG, Pradhan DS (1989) Evidence for the presence of DNA primase in mitochondria of Saccharomyces cerevisiae. Biochem Biophys Res Commun 160:525–534

    Article  CAS  PubMed  Google Scholar 

  • Deschauer M, Kiefer R, Blakely EL, He L, Zierz S, Turnbull DM, Taylor RW (2003) A novel Twinkle gene mutation in autosomal dominant progressive external ophthalmoplegia. Neuromuscul Disord 13:568–572

    Article  PubMed  Google Scholar 

  • Douglas SE (1998) Plastid evolution: origins, diversity, trends. Curr Opin Genet Dev 8:655–661

    Article  CAS  PubMed  Google Scholar 

  • Fahrni JF, Bolivar I, Berney C, Nassonova E, Smirnov A, Pawlowski J (2003) Phylogeny of lobose amoebae based on actin and small-subunit ribosomal RNA genes. J Mol Evol 20:1881–1886

    Article  CAS  Google Scholar 

  • Filée J, Forterre P, Sen-Lin T, Laurent J (2002) Evolution of DNA polymerase families: evidences for multiple gene exchange between cellular and viral proteins. J Mol Evol 54:763–773

    Article  PubMed  Google Scholar 

  • Fölsch H, Gaume B, Brunner M, Neupert W, Stuart RA. (1998) C- to N-terminal translocation of preproteins into mitochondria. EMBO J 17:6508–6515

    Article  PubMed  Google Scholar 

  • Grams J, Morris JC, Drew ME, Wang Z, Englund PT, Hajduk SL (2002) A trypanosome mitochondrial RNA polymerase is required for transcription and replication. J Biol Chem 277:16952–16959

    Article  CAS  PubMed  Google Scholar 

  • Guo S, Tabor S, Richardson CC (1999) The linker region between the helicase and primase domains of the bacteriophage T7 gene 4 protein is critical for hexamer formation. J Biol Chem 274:30303–30309

    Article  CAS  PubMed  Google Scholar 

  • Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98

    CAS  Google Scholar 

  • Harper JT, Waanders E, Keeling PJ (2005) On the monophyly of chromalveolates using a six–protein phylogeny of eukaryotes. Int J Syst Evol Microbiol 55:487–496

    Article  CAS  PubMed  Google Scholar 

  • Hedtke B, Börner T, Weihe A (2000) One RNA polymerase serving two genomes. EMBO Rep 1:435–440

    Article  CAS  PubMed  Google Scholar 

  • Hudson G, Deschauer M, Busse K, Zierz S, Chinnery PF (2005) Sensory ataxic neuropathy due to a novel C10Orf2 mutation with probable germline mosaicism. Neurology 64:371–373

    CAS  PubMed  Google Scholar 

  • Ilyina TV, Gorbalenya AE, Koonin EV (1992) Organization and evolution of bacterial and bacteriophage primase-helicase systems. J Mol Evol 34:351–357

    Article  CAS  PubMed  Google Scholar 

  • Johnson CE, Englund PT (1999) A refined localization of the mitochondrial DNA primase in Crithidia fasciculata. Mol Biochem Parasitol 102:205–208

    Article  CAS  PubMed  Google Scholar 

  • Kato M, Frick DN, Lee J, Tabor S, Richardson CC, Ellenberger T (2001) A complex of the bacteriophage T7 primase-helicase and DNA polymerase directs primer utilization. J Biol Chem 276:21809–21820

    Article  CAS  PubMed  Google Scholar 

  • Kato M, Ito T, Wagner G, Richardson CC, Ellenberger T (2003) Modular architecture of the bacteriophage T7 primase couples RNA primer synthesis to DNA synthesis. Mol Cell 11:1349–1360

    Article  CAS  PubMed  Google Scholar 

  • Korhonen JA, Gaspari M, Falkenberg M (2003) TWINKLE has 5′ → 3′ DNA helicase activity and is specifically stimulated by mitochondrial single-stranded DNA-binding protein. J Biol Chem 278:48627–48632

    Article  CAS  PubMed  Google Scholar 

  • Korhonen JA, Pham XH, Pellegrini M, Falkenberg M (2004) Reconstitution of a minimal mtDNA replisome in vitro. EMBO J 23:2423–2429

    Article  CAS  PubMed  Google Scholar 

  • Krishna SS, Majumdar I, Grishin NV (2003) Structural classification of zinc fingers: survey and summary. Nucleic Acids Res 31:532–550

    Article  CAS  PubMed  Google Scholar 

  • Kusakabe T, Richardson CC (1996) The role of the zinc motif in sequence recognition by DNA primases. J Biol Chem 271:19563–19570

    Article  CAS  PubMed  Google Scholar 

  • Lahaye A, Stahl H, Thines-Sempoux D, Foury F (1991) PIF1: a DNA helicase in yeast mitochondria. EMBO J 10:997–1007

    CAS  PubMed  Google Scholar 

  • Lang BF, O’Kelly C, Nerad T, Gray MW, Burger G (2002) The closest unicellular relatives of animals. Curr Biol 12:1773–1778

    Article  CAS  PubMed  Google Scholar 

  • Lecrenier N, Foury F (2000) New features of mitochondrial DNA replication system in yeast and man. Gene 246:37–48

    Article  CAS  PubMed  Google Scholar 

  • Lee CM, Sedman J, Neupert W, Stuart RA (1999) The DNA helicase, Hmi1p, is transported into mitochondria by a C-terminal cleavable targeting signal. J Biol Chem 274:20937–20942

    Article  CAS  PubMed  Google Scholar 

  • Lee SJ, Richardson CC (2001) Essential lysine residues in the RNA polymerase domain of the gene 4 primase-helicase of bacteriophage T7 J Biol Chem 276:49419–49426

    Article  CAS  PubMed  Google Scholar 

  • Lee SJ, Richardson CC (2005) Acidic residues in the nucleotide binding site of the bacteriophage T7 DNA primase. J Biol Chem 280:26984–26991

    Article  CAS  PubMed  Google Scholar 

  • Leipe DD, Aravind L, Grishin NV, Koonin EV (2000) The bacterial replicative helicase DnaB evolved from a RecA duplication. Genome Res 10:5–16

    CAS  PubMed  Google Scholar 

  • Lewis S, Hutchison W, Thyagarajan D, Dahl HH (2002) Clinical and molecular features of adPEO due to mutations in the Twinkle gene. J Neurol Sci 201:39–44

    Article  CAS  PubMed  Google Scholar 

  • Li C, Englund PT (1997) A mitochondrial DNA primase from the trypanosomatid Crithidia fasciculata. J Biol Chem 272:20787–20792

    Article  CAS  PubMed  Google Scholar 

  • Li FY, Tariq M, Croxen R, Morten K, Squier W, Newsom-Davis J, Beeson D, Larsson C (1999) Mapping of autosomal dominant progressive external ophthalmoplegia to a 7-cM critical region on 10q24 Neurology 53:1265–1271

    CAS  PubMed  Google Scholar 

  • Lu YB, Ratnakar PV, Mohanty BK, Bastia D (1996) Direct physical interaction between DnaG primase and DnaB helicase of Escherichia coli is necessary for optimal synthesis of primer RNA. Proc Natl Acad Sci USA 93:12902–12907

    Article  CAS  PubMed  Google Scholar 

  • Morris JC, Drew ME, Klingbeil MM, Motyka SA, Saxowsky TT, Wang Z, Englund PT (2001) Replication of kinetoplast DNA: an update for the new millennium. Int J Parasitol 31:453–458

    Article  CAS  PubMed  Google Scholar 

  • Nikolaev SI, Berney C, Fahrni JF, Bolivar I, Polet S, Mylnikov AP, Aleshin VV, Petrov NB, Pawlowski J (2004) The twilight of Heliozoa and rise of Rhizaria, an emerging supergroup of amoeboid eukaryotes. Proc Natl Acad Sci USA 101:8066–8071

    Article  CAS  PubMed  Google Scholar 

  • Sarkar G, Turner RT, Bolander ME (1993) Restriction-site PCR: a direct method of unknown sequence retrieval adjacent to a known locus by using universal primers. PCR Methods Appl 2:318–322

    CAS  PubMed  Google Scholar 

  • Sawaya MR, Guo S, Tabor S, Richardson CC, Ellenberger T (1999) Crystal structure of the helicase domain from the replicative helicase-primase of bacteriophage T7. Cell 99:167–177

    Article  CAS  PubMed  Google Scholar 

  • Sedman T, Kuusk S, Kivi S, Sedman J (2000) A DNA helicase required for maintenance of the functional mitochondrial genome in Saccharomyces cerevisiae. Mol Cell Biol 20:1816–1824

    Article  CAS  PubMed  Google Scholar 

  • Seow F, Sato S, Janssen CS, Riehle MO, Mukhopadhyay A, Phillips RS, Wilson RJ, Barrett MP (2005) The plastidic DNA replication enzyme complex of Plasmodium falciparum. Mol Biochem Parasitol 141:145–153

    Article  CAS  PubMed  Google Scholar 

  • Simpson AGB (2003) Cytoskeletal organization, phylogenetic affinities and systematics in the contentious taxon Excavata (Eukaryota). Int J Syst Evol Microbiol 53:1759–1777

    Article  PubMed  Google Scholar 

  • Simpson AGB, Roger AJ (2004) The real ‘kingdoms’ of eukaryotes. Curr Biol 14:R693–R696

    Article  CAS  PubMed  Google Scholar 

  • Singleton MR, Sawaya MR, Ellenberger T, Wigley DB (2000) Crystal structure of T7 gene 4 ring helicase indicates a mechanism for sequential hydrolysis of nucleotides. Cell 101:589–600

    Article  CAS  PubMed  Google Scholar 

  • Soultanas P (2005) The bacterial helicase-primase interaction: a common structural/functional module. Structure 13:839–844

    Article  CAS  PubMed  Google Scholar 

  • Spelbrink JN, Li F-Y, Tiranti V, Nikali K, Yuan Q-P, Tariq M, Wanrooij S, Garrido N, Comi G, Morandi L, Santoro L, Toscano A, Fabrizi G-M, Somer H, Croxen R, Beeson D, Poulton J, Suomalainen A, Jacobs HT, Zeviani M, Larsson C (2001) Human mitochondrial DNA deletions associated with mutations in the gene encoding Twinkle, a phage T7 gene 4-like protein localized in mitochondria. Nat Genet 28:223–231

    Article  CAS  PubMed  Google Scholar 

  • Steitz TA (1998) A mechanism for all polymerases. Nature 391:231–232

    Article  CAS  PubMed  Google Scholar 

  • Stoltzfus A, Logsdon JM Jr, Palmer JD, Doolittle WF (1997) Intron “sliding” and the diversity of intron positions. Proc Natl Acad Sci USA 94:10739–10744

    Article  CAS  PubMed  Google Scholar 

  • Sverdlov AV, Babenko VN, Rogozin IB, Koonin EV (2004) Preferential loss and gain of introns in 3′ portions of genes suggests a reverse-transcription mechanism of intron insertion. Gene 338:85–91

    Article  CAS  PubMed  Google Scholar 

  • Thirlway J, Turner IJ, Gibson CT, Gardiner L, Brady K, Allen S, Roberts CJ, Soultanas P (2004) DnaG interacts with a linker region that joins the N- and C-domains of DnaB and induces the formation of 3-fold symmetric rings. Nucleic Acids Res 32:2977–2986

    Article  CAS  PubMed  Google Scholar 

  • Toth EA, Li Y, Sawaya MR, Cheng Y, Ellenberger T (2003) The crystal structure of the bifunctional primase–helicase of bacteriophage T7 Mol Cell 12:1113–1123

    Article  CAS  PubMed  Google Scholar 

  • Tougu K, Peng H, Marians KJ (1994) Identification of a domain of Escherichia coli primase required for functional interaction with the DnaB helicase at the replication fork. J Biol Chem 269:4675–4682

    CAS  PubMed  Google Scholar 

  • Tyynismaa H, Sembongi H, Bokori-Brown M, Granycome C, Ashley N, Poulton J, Jalanko A, Spelbrink JN, Holt IJ, Suomalainen A (2004) Twinkle helicase is essential for mtDNA maintenance and regulates mtDNA copy number. Hum Mol Genet 13:3219–3227

    Article  CAS  PubMed  Google Scholar 

  • Van Goethem G, Löfgren A, Dermaut B, Ceuterick C, Martin J-J, Van Broeckhoven C (2003) Digenic progressive external ophthalmoplegia in a sporadic patient: recessive mutations in POLG and C10orf2/Twinkle. Hum Mutat 22:175–176

    Article  PubMed  Google Scholar 

  • Versalovic J, Lupski JR (1993) The Haemophilus influenzae dnaG sequence and conserved bacterial primase motifs. Gene 136:281–286

    Article  CAS  PubMed  Google Scholar 

  • Vishwanatha JK, Baril EF (1986) Resolution and purification of free primase activity from the DNA primase–polymerase α complex of HeLa cells. Nucleic Acids Res 14:8467–8487

    CAS  PubMed  Google Scholar 

  • Weber KL, Bolander ME, Sarkar G (1998) Rapid acquisition of unknown DNA sequence adjacent to a known segment by multiplex restriction site PCR. Biotechniques 25:415–419

    CAS  PubMed  Google Scholar 

  • Wong TW, Clayton DA (1985) Isolation and characterization of a DNA primase from human mitochondria. J Biol Chem 260:11530–11535

    CAS  PubMed  Google Scholar 

  • Zettler LAA, Nerad TA, O’Kelly CJ, Sogin ML (2001) The nucleariid amoebae: more protists at the animal-fungal boundary. J Eukaryot Microbiol 48:293–297

    Article  PubMed  Google Scholar 

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Acknowledgments

Twinkle sequences generated under the auspices of the Protist EST Program were generously provided by R.W. Lee, Dalhousie University (P. wickerhamii), and B.F. Lang, Université de Montréal (J. bahamiensis, M. jakobiformis, S. ecuadoriensis). Preliminary sequence data for Tetrahymena thermophila were obtained from The Institute for Genomic Research (TIGR) web site, at http://www.tigr.org. We are grateful to one of the reviewers of this manuscript for pointing out the Twinkle-like helicase sequence in the recently released Rhizopus oryzae genome data. We thank M. Dlutek for performing DNA sequencing. This work was supported by funding to M.W.G. from the Canadian Institutes of Health Research (Operating Grant MOP-4355) and Genome Canada through Genome Atlantic and the Atlantic Innovation Fund (Protist EST Program). M.W.G. is pleased to acknowledge salary support from the Canada Research Chairs Program and the Canadian Institute for Advanced Research.

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  1. Program in Evolutionary Biology, Canadian Institute for Advanced Research, and Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada

    Timothy E. Shutt & Michael W. Gray

  2. Room 8F-2, Department of Biochemistry and Molecular Biology, Sir Charles Tupper Medical Building, Dalhousie University, 5850 College Street, Halifax, NS, Canada, B3H 1X5

    Michael W. Gray

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  1. Timothy E. Shutt
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Correspondence to Michael W. Gray.

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Shutt, T.E., Gray, M.W. Twinkle, the Mitochondrial Replicative DNA Helicase, Is Widespread in the Eukaryotic Radiation and May Also Be the Mitochondrial DNA Primase in Most Eukaryotes. J Mol Evol 62, 588–599 (2006). https://doi.org/10.1007/s00239-005-0162-8

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