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Human mitochondrial DNA deletions associated with mutations in the gene encoding Twinkle, a phage T7 gene 4-like protein localized in mitochondria

A Correction to this article was published on 01 September 2001

Abstract

The gene products involved in mammalian mitochondrial DNA (mtDNA) maintenance and organization remain largely unknown. We report here a novel mitochondrial protein, Twinkle, with structural similarity to phage T7 gene 4 primase/helicase and other hexameric ring helicases. Twinkle colocalizes with mtDNA in mitochondrial nucleoids. Screening of the gene encoding Twinkle in individuals with autosomal dominant progressive external ophthalmoplegia (adPEO), associated with multiple mtDNA deletions, identified 11 different coding-region mutations co-segregating with the disorder in 12 adPEO pedigrees of various ethnic origins. The mutations cluster in a region of the protein proposed to be involved in subunit interactions. The function of Twinkle is inferred to be critical for lifetime maintenance of human mtDNA integrity.

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Figure 1: C10orf2 gene structure and mRNA expression in human tissues.
Figure 2: Multiple sequence alignment of C10orf2 protein with T7 gp4 and several eukaryotic homologs.
Figure 3: Subcellular localization of C10orf2-EGFP fusion proteins.
Figure 4: Colocalization of Twinkle-EGFP with EtBr-stained mtDNA.
Figure 5: Enhancement of mitochondrial DNA helicase activity by transient expression of Twinkle tagged (or untagged).
Figure 6: Twinkle mutations and segregation analysis in large adPEO pedigrees In each pedigree mutations were initially identified by direct sequencing of genomic DNA from at least one individual.
Figure 7: Multimerization of Twinkle and its variants.

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References

  1. Foury, F. Cloning and sequencing of the nuclear gene MIP 1 encoding the catalytic subunit of the yeast mitochondrial DNA polymerase. J. Biol. Chem. 264, 20552–20560 (1989).

    CAS  PubMed  Google Scholar 

  2. Iyengar, B., Roote, J. & Campos, A.R. The tamas gene, identified as a mutation that disrupts larval behavior in Drosophila melanogaster, codes for the mitochondrial DNA polymerase catalytic subunit (DNApol-gamma125). Genetics 153, 1809–1824 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Lim, S.E., Longley, M.J. & Copeland, W.C. The mitochondrial p55 accessory subunit of human DNA polymerase gamma enhances DNA binding, promotes processive DNA synthesis, and confers N-ethylmaleimide resistance. J. Biol. Chem. 274, 38197–38203 (1999).

    Article  CAS  Google Scholar 

  4. Clayton, D.A. Replication of animal mitochondrial DNA. Cell 28, 693–705 (1982).

    Article  CAS  Google Scholar 

  5. Holt, I.J., Lorimer, H.E. & Jacobs, H.T. Coupled leading- and lagging-strand synthesis of mammalian mitochondrial DNA. Cell 100, 515–524 (2000).

    Article  CAS  Google Scholar 

  6. Lecrenier, N., Van Der Bruggen, P. & Foury, F. Mitochondrial DNA polymerases from yeast to man: a new family of polymerases. Gene 185, 147–152 (1997).

    Article  CAS  Google Scholar 

  7. Kornberg, A. & Baker, T.A. DNA Replication (W.H. Freeman and Company, New York, 1992).

    Google Scholar 

  8. Tiranti, V. et al. Identification of the gene encoding the human mitochondrial RNA polymerase (h-mtRPOL) by cyberscreening of the Expressed Sequence Tags database. Hum. Mol. Genet. 6, 615–625 (1997).

    Article  CAS  Google Scholar 

  9. Zeviani, M. et al. An autosomal dominant disorder with multiple deletions of mitochondrial DNA starting at the D-loop region. Nature 339, 309–311 (1989).

    Article  CAS  Google Scholar 

  10. Zeviani, M. et al. Nucleus-driven multiple large-scale deletions of the human mitochondrial genome: A new autosomal dominant disease. Am. J. Hum. Genet. 47, 904–914 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Suomalainen, A. et al. Multiple deletions of mitochondrial DNA in several tissues of a patient with severe retarded depression and familial progressive external ophthalmoplegia. J. Clin. Invest. 90, 61–66 (1992).

    Article  CAS  Google Scholar 

  12. Melberg, A., Lundberg, P.O., Henriksson, K.G., Olsson, Y. & Stalberg, E. Muscle-nerve involvement in autosomal dominant progressive external ophthalmoplegia with hypogonadism. Muscle Nerve 19, 751–757 (1996).

    Article  CAS  Google Scholar 

  13. Li, F.Y. et al. Mapping of autosomal dominant progressive external ophthalmoplegia to a 7-cM critical region on 10q24. Neurology 53, 1265–1271 (1999).

    Article  CAS  Google Scholar 

  14. Moslemi, A.-R., Melberg, A., Holme, E. & Oldfors, A. Clonal expansion of mitochondrial DNA with multiple deletions in autosomal dominant progressive external ophthalmoplegia. Ann. Neurol. 40, 707–713 (1996).

    Article  CAS  Google Scholar 

  15. Santorelli, F.M. et al. Multiple mtDNA deletions: clinical and molecular correlations. J. Inherit. Metab. Dis. 23, 155–161 (2000).

    Article  CAS  Google Scholar 

  16. Bohlega, S. et al. Multiple mitochondrial DNA deletions associated with autosomal recessive ophthalmoplegia and severe cardiomyopathy. Neurology 46, 1329–1334 (1996).

    Article  CAS  Google Scholar 

  17. Suomalainen, A. et al. An autosomal locus predisposing to deletions of mitochondrial DNA. Nature Genet. 9, 146–151 (1995).

    Article  CAS  Google Scholar 

  18. Kaukonen, J. et al. A third locus predisposing to multiple deletions of mtDNA in autosomal dominant progressive external ophthalmoplegia Am. J. Hum. Genet. 65, 256–261 (1999).

    Article  CAS  Google Scholar 

  19. Kaukonen, J. et al. Role of adenine nucleotide translocator 1 in mtDNA maintenance. Science 289, 782–785 (2000).

    Article  CAS  Google Scholar 

  20. Nishino, I., Spinazzola, A. & Hirano, M. Thymidine phosphorylase gene mutations in MNGIE, a human mitochondrial disorder. Science 283, 689–692 (1999).

    Article  CAS  Google Scholar 

  21. Leipe, D.D., Aravind, L., Grishin, N.V. & Koonin, E.V. The bacterial replicative helicase DnaB evolved from a RecA duplication. Genome Res. 10, 5–16 (2000).

    CAS  PubMed  Google Scholar 

  22. Lennon, G., Auffray, C., Polymeropoulos, M. & Soares, M.B. The I.M.A.G.E. Consortium: an integrated molecular analysis of genomes and their expression. Genomics 33, 151–152 (1996).

    Article  CAS  Google Scholar 

  23. Bird, L.E., Hakansson, K., Pan, H. & Wigley, D.B. Characterization and crystallization of the helicase domain of bacteriophage T7 gene 4 protein. Nucleic Acids Res. 25, 2620–2626 (1997).

    Article  CAS  Google Scholar 

  24. Hayashi, J.-I., Takemitsu, M., Goto, Y.-I. & Nonaka, I. Human mitochondria and mitochondrial genome function as a single dynamic cellular unit. J. Cell Biol. 125, 43–50 (1994).

    Article  CAS  Google Scholar 

  25. Davis, A.F. & Clayton, D.A. In situ localization of mitochondrial DNA replication in intact mammalian cells. J. Cell Biol. 135, 883–893 (1996).

    Article  CAS  Google Scholar 

  26. Satoh, M. & Kuroiwa, T. Organization of multiple nucleoids and DNA molecules in mitochondria of a human cell. Exp. Cell Res. 196, 137–140 (1991).

    Article  CAS  Google Scholar 

  27. Spelbrink, J.N. et al. In vivo functional analysis of the human mitochondrial DNA polymerase POLG expressed in cultured human cells. J. Biol. Chem. 275, 24818–24828 (2000).

    Article  CAS  Google Scholar 

  28. Washington, M.T., Rosenberg, A.H., Griffin, K., Studier, F.W. & Patel, S.S. Biochemical analysis of mutant T7 primase/helicase proteins defective in DNA binding, nucleotide hydrolysis, and the coupling of hydrolysis with DNA unwinding. J. Biol. Chem. 271, 26825–26834 (1996).

    Article  CAS  Google Scholar 

  29. Hehman, G.L. & Hauswirth, W.W. DNA helicase from mammalian mitochondria. Proc. Natl. Acad. Sci. USA 89, 8562–8566 (1992).

    Article  CAS  Google Scholar 

  30. Li, F. et al. Characterization of a novel human putative mitochondrial transporter homologous to the yeast mitochondrial RNA splicing proteins 3 and 4. FEBS Lett. 494, 79–84 (2001).

    Article  CAS  Google Scholar 

  31. Guo, S., Tabor, S. & Richardson, C.C. 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 (1999).

    Article  CAS  Google Scholar 

  32. Singleton, M.R., Sawaya, M.R., Ellenberger, T. & Wigley, D.B. Crystal structure of T7 gene 4 ring helicase indicates a mechanism for sequential hydrolysis of nucleotides. Cell 101, 589–600 (2000).

    Article  CAS  Google Scholar 

  33. Patel, S.S. & Hingorani, M.M. Oligomeric structure of bacteriophage T7 DNA primase/helicase proteins. J. Biol. Chem. 268, 10668–10675 (1993).

    CAS  PubMed  Google Scholar 

  34. Hingorani, M.M. & Patel, S.S. Cooperative interactions of nucleotide ligands are linked to oligomerization and DNA binding in bacteriophage T7 gene 4 helicases. Biochemistry 35, 2218–2228 (1996).

    Article  CAS  Google Scholar 

  35. Ilyina, T.V., Gorbalenya, A.E. & Koonin, E.V. Organization and evolution of bacterial and bacteriophage primase- helicase systems. J. Mol. Evol. 34, 351–357 (1992).

    Article  CAS  Google Scholar 

  36. Miyakawa, I., Sando, N., Kawano, S., Nakamura, S. & Kuroiwa, T. Isolation of morphologically intact mitochondrial nucleoids from the yeast, Saccharomyces cerevisiae. J. Cell Sci. 88, 431–439 (1987).

    CAS  PubMed  Google Scholar 

  37. Kuroiwa, T. et al. The division apparatus of plastids and mitochondria. Int. Rev. Cytol. 181, 1–41 (1998).

    Article  CAS  Google Scholar 

  38. Kaufman, B.A. et al. In organelle formaldehyde crosslinking of proteins to mtDNA: identification of bifunctional proteins. Proc. Natl. Acad. Sci. USA 97, 7772–7777 (2000).

    Article  CAS  Google Scholar 

  39. Robin, E.D. & Wong, R. Mitochondrial DNA molecules and virtual number of mitochondria per cell in mammalian cells. J. Cell. Physiol. 136, 507–513 (1988).

    Article  CAS  Google Scholar 

  40. Zhang, H. et al. Quantitation of mitochondrial DNA in human lymphoblasts by a competitive polymerase chain reaction method: application to the study of inhibitors of mitochondrial DNA content. Mol. Pharmacol. 46, 1063–1069 (1994).

    CAS  PubMed  Google Scholar 

  41. Doublie, S., Tabor, S., Long, A.M., Richardson, C.C. & Ellenberger, T. Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 A resolution Nature 391, 251–258 (1998).

    Article  CAS  Google Scholar 

  42. Sawaya, M.R., Guo, S., Tabor, S., Richardson, C.C. & Ellenberger, T. Crystal structure of the helicase domain from the replicative helicase-primase of bacteriophage T7. Cell 99, 167–177 (1999).

    Article  CAS  Google Scholar 

  43. Kajander, O.A. et al. Human mtDNA sublimons resemble rearranged mitochondrial genomes found in pathological states. Hum. Mol. Genet. 9, 2821–2835 (2000).

    Article  CAS  Google Scholar 

  44. Van Goethem, G., Löfgren, A., Dermaut, B., Martin, J-J. & Van Broeckhoven, C. Mutation of POLG is associated with autosomal dominant progressive external opthalmoplegia with multiple mtDNA deletions. Nature Genet. 28, 211–212 (2001).

    Article  CAS  Google Scholar 

  45. Yu, C.E. et al. Positional cloning of the Werner's syndrome gene Science 272, 258–262 (1996).

    Article  CAS  Google Scholar 

  46. Sinclair, D.A. & Guarente, L. Extrachromosomal rDNA circles—a cause of aging in yeast. Cell 91, 1033–1042 (1997).

    Article  CAS  Google Scholar 

  47. Suomalainen, A. et al. Autosomal dominant progressive external ophthalmoplegia with multiple deletions of mtDNA: clinical, biochemical, and molecular genetic features of the 10q-linked disease. Neurology 48, 1244–1253 (1997).

    Article  CAS  Google Scholar 

  48. Davis, L.G., Dibner, M.D. & Battey, J.F. Basic Methods in Molecular Biology (Elsevier Science, New York, 1986).

    Google Scholar 

  49. Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685 (1970).

    Article  CAS  Google Scholar 

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Acknowledgements

Financial support was provided by the Academy of Finland, Tampere University Hospital Medical Research Fund, Juselius Foundation, Finnish Cultural Foundation, Swedish Medical Research Council, Torsten and Ragnar Söderberg Memory Foundations, Telethon grant 1180, “Ricerca Finalizzata” grant ICS030.3/RF98.37, a Pier Franco and Luisa Mariani Foundation grant to M.Z. and the EU BIOMED2 programme. R.C. and D.B. were supported by the Medical Research Council (U.K.) and the Muscular Dystrophy Campaign/Myasthenia Gravis Association. J.P. is a Royal Society University Research Fellow. We thank Anja Rovio for technical assistance, Leena Peltonen for sharing her expertise, Sanna Lehtinen for expression studies of Twinkle-EGFP in A549-derived cells and Dale Wigley and Ian Holt for many useful discussions.

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Spelbrink, J., Li, FY., Tiranti, V. et al. 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 (2001). https://doi.org/10.1038/90058

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