Advertisement

Analysis of Replicating Mitochondrial DNA by In Organello Labeling and Two-Dimensional Agarose Gel Electrophoresis

  • Ian J. HoltEmail author
  • Lawrence Kazak
  • Aurelio Reyes
  • Stuart R. Wood
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1351)

Abstract

Our understanding of the mechanisms of DNA replication in a broad range of organisms and viruses has benefited from the application of two-dimensional agarose gel electrophoresis (2D-AGE). The method resolves DNA molecules on the basis of size and shape and is technically straightforward. 2D-AGE sparked controversy in the field of mitochondria when it revealed replicating molecules with lengthy tracts of RNA, a phenomenon never before reported in nature. More recently, radioisotope labeling of the DNA in the mitochondria has been coupled with 2D-AGE. In its first application, this procedure helped to delineate the “bootlace mechanism of mitochondrial DNA replication,” in which processed mitochondrial transcripts are hybridized to the lagging strand template at the replication fork as the leading DNA strand is synthesized. This chapter provides details of the method, how it has been applied to date and concludes with some potential future applications of the technique.

Key words

DNA replication DNA labeling Mitochondrial DNA Two-dimensional agarose gel electrophoresis 2D-AGE Replication intermediates Humans Mammals 

Notes

Acknowledgements

We thank Drs. Pohjoismäki and Jacobs for the EM image of the replicating mitochondrial DNA molecule, and the Medical Research Council for funding.

References

  1. 1.
    Holt IJ, Harding AE, Morgan-Hughes JA (1988) Deletions of muscle mitochondrial DNA in patients with mitochondrial myopathies. Nature 331:717–719CrossRefPubMedGoogle Scholar
  2. 2.
    Spelbrink JN, Li FY, Tiranti V, Nikali K, Yuan QP, Tariq M, Wanrooij S, Garrido N, Comi G, Morandi L, Santoro L, Toscano A, Fabrizi GM, 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 28:223–231CrossRefPubMedGoogle Scholar
  3. 3.
    Van Goethem G, Dermaut B, Lofgren A, Martin JJ, Van Broeckhoven C (2001) Mutation of POLG is associated with progressive external ophthalmoplegia characterized by mtDNA deletions. Nat Genet 28:211–212CrossRefPubMedGoogle Scholar
  4. 4.
    Goto Y, Nonaka I, Horai S (1990) A mutation in the tRNA(Leu)(UUR) gene associated with the MELAS subgroup of mitochondrial encephalomyopathies. Nature 348:651–653CrossRefPubMedGoogle Scholar
  5. 5.
    Shoffner JM, Lott MT, Lezza AM, Seibel P, Ballinger SW, Wallace DC (1990) Myoclonic epilepsy and ragged-red fiber disease (MERRF) is associated with a mitochondrial DNA tRNA(Lys) mutation. Cell 61:931–937CrossRefPubMedGoogle Scholar
  6. 6.
    Kraytsberg Y, Kudryavtseva E, McKee AC, Geula C, Kowall NW, Khrapko K (2006) Mitochondrial DNA deletions are abundant and cause functional impairment in aged human substantia nigra neurons. Nat Genet 38:518–520CrossRefPubMedGoogle Scholar
  7. 7.
    Bender A, Krishnan KJ, Morris CM, Taylor GA, Reeve AK, Perry RH, Jaros E, Hersheson JS, Betts J, Klopstock T, Taylor RW, Turnbull DM (2006) High levels of mitochondrial DNA deletions in substantia nigra neurons in aging and Parkinson disease. Nat Genet 38:515–517CrossRefPubMedGoogle Scholar
  8. 8.
    Herbst A, Pak JW, McKenzie D, Bua E, Bassiouni M, Aiken JM (2007) Accumulation of mitochondrial DNA deletion mutations in aged muscle fibers: evidence for a causal role in muscle fiber loss. J Gerontol A Biol Sci Med Sci 62:235–245CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Kowald A, Dawson M, Kirkwood TB (2014) Mitochondrial mutations and ageing: Can mitochondrial deletion mutants accumulate via a size based replication advantage? J Theor Biol 340:111–118CrossRefPubMedGoogle Scholar
  10. 10.
    Nguyen KV, Sharief FS, Chan SS, Copeland WC, Naviaux RK (2006) Molecular diagnosis of Alpers syndrome. J Hepatol 45:108–116CrossRefPubMedGoogle Scholar
  11. 11.
    Trifunovic A, Wredenberg A, Falkenberg M, Spelbrink JN, Rovio AT, Bruder CE, Bohlooly YM, Gidlof S, Oldfors A, Wibom R, Tornell J, Jacobs HT, Larsson NG (2004) Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature 429:417–423CrossRefPubMedGoogle Scholar
  12. 12.
    Bell L, Byers B (1983) Separation of branched from linear DNA by two-dimensional gel electrophoresis. Anal Biochem 130:527–535CrossRefPubMedGoogle Scholar
  13. 13.
    Brewer BJ, Fangman WL (1987) The localization of replication origins on ARS plasmids in S. cerevisiae. Cell 51:463–471CrossRefPubMedGoogle Scholar
  14. 14.
    Brewer BJ, Fangman WL (1988) A replication fork barrier at the 3′ end of yeast ribosomal RNA genes. Cell 55:637–643CrossRefPubMedGoogle Scholar
  15. 15.
    Brewer BJ, Fangman WL (1991) Mapping replication origins in yeast chromosomes. Bioessays 13:317–322CrossRefPubMedGoogle Scholar
  16. 16.
    Brun C, Dijkwel PA, Little RD, Hamlin JL, Schildkraut CL, Huberman JA (1995) Yeast and mammalian replication intermediates migrate similarly in two-dimensional gels. Chromosoma 104:92–102CrossRefPubMedGoogle Scholar
  17. 17.
    Dijkwel PA, Vaughn JP, Hamlin JL (1994) Replication initiation sites are distributed widely in the amplified CHO dihydrofolate reductase domain. Nucleic Acids Res 22:4989–4996CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    van Brabant AJ, Hunt SY, Fangman WL, Brewer BJ (1998) Identifying sites of replication initiation in yeast chromosomes: looking for origins in all the right places. Electrophoresis 19:1239–1246CrossRefPubMedGoogle Scholar
  19. 19.
    Brewer BJ, Lockshon D, Fangman WL (1992) The arrest of replication forks in the rDNA of yeast occurs independently of transcription. Cell 71:267–276CrossRefPubMedGoogle Scholar
  20. 20.
    Viguera E, Hernandez P, Krimer DB, Boistov AS, Lurz R, Alonso JC, Schvartzman JB (1996) The ColE1 unidirectional origin acts as a polar replication fork pausing site. J Biol Chem 271:22414–22421CrossRefPubMedGoogle Scholar
  21. 21.
    Friedman KL, Brewer BJ (1995) Analysis of replication intermediates by two-dimensional agarose gel electrophoresis. Methods Enzymol 262:613–627CrossRefPubMedGoogle Scholar
  22. 22.
    Reyes A, Yang MY, Bowmaker M, Holt IJ (2005) Bidirectional replication initiates at sites throughout the mitochondrial genome of birds. J Biol Chem 280:3242–3250CrossRefPubMedGoogle Scholar
  23. 23.
    Belanger KG, Mirzayan C, Kreuzer HE, Alberts BM, Kreuzer KN (1996) Two-dimensional gel analysis of rolling circle replication in the presence and absence of bacteriophage T4 primase. Nucleic Acids Res 24:2166–2175CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Han Z, Stachow C (1994) Analysis of Schizosaccharomyces pombe mitochondrial DNA replication by two dimensional gel electrophoresis. Chromosoma 103:162–170PubMedGoogle Scholar
  25. 25.
    Kamath S, Leffak M (2001) Multiple sites of replication initiation in the human beta-globin gene locus. Nucleic Acids Res 29:809–817CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Lunyak VV, Ezrokhi M, Smith HS, Gerbi SA (2002) Developmental changes in the Sciara II/9A initiation zone for DNA replication. Mol Cell Biol 22:8426–8437CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Preiser PR, Wilson RJ, Moore PW, McCready S, Hajibagheri MA, Blight KJ, Strath M, Williamson DH (1996) Recombination associated with replication of malarial mitochondrial DNA. EMBO J 15:684–693PubMedPubMedCentralGoogle Scholar
  28. 28.
    Mesner LD, Li X, Dijkwel PA, Hamlin JL (2003) The dihydrofolate reductase origin of replication does not contain any nonredundant genetic elements required for origin activity. Mol Cell Biol 23:804–814CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Dijkwel PA, Hamlin JL (1997) Mapping replication origins by neutral/neutral two-dimensional gel electrophoresis. Methods 13:235–245CrossRefPubMedGoogle Scholar
  30. 30.
    Kuzminov A, Schabtach E, Stahl FW (1997) Study of plasmid replication in Escherichia coli with a combination of 2D gel electrophoresis and electron microscopy. J Mol Biol 268:1–7CrossRefPubMedGoogle Scholar
  31. 31.
    Linskens MH, Huberman JA (1990) Ambiguities in results obtained with 2D gel replicon mapping techniques. Nucleic Acids Res 18:647–652CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Little RD, Schildkraut CL (1995) Initiation of latent DNA replication in the Epstein-Barr virus genome can occur at sites other than the genetically defined origin. Mol Cell Biol 15:2893–2903CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Mayhook AG, Rinaldi AM, Jacobs HT (1992) Replication origins and pause sites in sea urchin mitochondrial DNA. Proc R Soc Lond B Biol Sci 248:85–94CrossRefGoogle Scholar
  34. 34.
    Robinson NP (2013) Analysis of branched DNA replication and recombination intermediates from prokaryotic cells by two-dimensional (2D) native-native agarose gel electrophoresis. Methods Mol Biol 1054:45–61CrossRefPubMedGoogle Scholar
  35. 35.
    Schvartzman JB, Martinez-Robles ML, Hernandez P (1993) The migration behaviour of DNA replicative intermediates containing an internal bubble analyzed by two-dimensional agarose gel electrophoresis. Nucleic Acids Res 21:5474–5479CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Bowmaker M, Yang MY, Yasukawa T, Reyes A, Jacobs HT, Huberman JA, Holt IJ (2003) Mammalian mitochondrial DNA replicates bidirectionally from an initiation zone. J Biol Chem 278:50961–50969CrossRefPubMedGoogle Scholar
  37. 37.
    Grossman LI, Watson R, Vinograd J (1973) The presence of ribonucleotides in mature closed-circular mitochondrial DNA. Proc Natl Acad Sci U S A 70:3339–3343CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Yang MY, Bowmaker M, Reyes A, Vergani L, Angeli P, Gringeri E, Jacobs HT, Holt IJ (2002) Biased incorporation of ribonucleotides on the mitochondrial L-strand accounts for apparent strand-asymmetric DNA replication. Cell 111:495–505CrossRefPubMedGoogle Scholar
  39. 39.
    Yasukawa T, Reyes A, Cluett TJ, Yang MY, Bowmaker M, Jacobs HT, Holt IJ (2006) Replication of vertebrate mitochondrial DNA entails transient ribonucleotide incorporation throughout the lagging strand. EMBO J 25:5358–5371CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Pohjoismaki JL, Holmes JB, Wood SR, Yang MY, Yasukawa T, Reyes A, Bailey LJ, Cluett TJ, Goffart S, Willcox S, Rigby RE, Jackson AP, Spelbrink JN, Griffith JD, Crouch RJ, Jacobs HT, Holt IJ (2010) Mammalian mitochondrial DNA replication intermediates are essentially duplex but contain extensive tracts of RNA/DNA hybrid. J Mol Biol 397:1144–1155CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Reyes A, Kazak L, Wood SR, Yasukawa T, Jacobs HT, Holt IJ (2013) Mitochondrial DNA replication proceeds via a “bootlace” mechanism involving the incorporation of processed transcripts. Nucleic Acids Res 41:5837–5850CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Miralles Fuste J, Shi Y, Wanrooij S, Zhu X, Jemt E, Persson O, Sabouri N, Gustafsson CM, Falkenberg M (2014) In vivo occupancy of mitochondrial single-stranded DNA binding protein supports the strand displacement mode of DNA replication. PLoS Genet 10:e1004832CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Holt IJ, Jacobs HT (2014) Unique features of DNA replication in mitochondria: a functional and evolutionary perspective. Bioessays 36:1024–1031CrossRefPubMedGoogle Scholar
  44. 44.
    Huberman JA (1997) Mapping replication origins, pause sites, and termini by neutral/alkaline two-dimensional gel electrophoresis. Methods 13:247–257CrossRefPubMedGoogle Scholar
  45. 45.
    Enriquez JA, Ramos J, Perez-Martos A, Lopez-Perez MJ, Montoya J (1994) Highly efficient DNA synthesis in isolated mitochondria from rat liver. Nucleic Acids Res 22:1861–1865CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Cantatore P, Loguercio Polosa P, Mustich A, Petruzzella V, Gadaleta MN (1988) Faithful and highly efficient RNA synthesis in isolated mitochondria from rat liver. Curr Genet 14:477–482CrossRefPubMedGoogle Scholar
  47. 47.
    Yasukawa T, Yang MY, Jacobs HT, Holt IJ (2005) A bidirectional origin of replication maps to the major noncoding region of human mitochondrial DNA. Mol Cell 18:651–662CrossRefPubMedGoogle Scholar
  48. 48.
    Holt IJ, Lorimer HE, Jacobs HT (2000) Coupled leading- and lagging-strand synthesis of mammalian mitochondrial DNA. Cell 100:515–524CrossRefPubMedGoogle Scholar
  49. 49.
    Pohjoismaki JL, Goffart S, Tyynismaa H, Willcox S, Ide T, Kang D, Suomalainen A, Karhunen PJ, Griffith JD, Holt IJ, Jacobs HT (2009) Human heart mitochondrial DNA is organized in complex catenated networks containing abundant four-way junctions and replication forks. J Biol Chem 284:21446–21457CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Gensler S, Weber K, Schmitt WE, Perez-Martos A, Enriquez JA, Montoya J, Wiesner RJ (2001) Mechanism of mammalian mitochondrial DNA replication: import of mitochondrial transcription factor A into isolated mitochondria stimulates 7S DNA synthesis. Nucleic Acids Res 29:3657–3663CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Wanrooij S, Goffart S, Pohjoismaki JL, Yasukawa T, Spelbrink JN (2007) Expression of catalytic mutants of the mtDNA helicase Twinkle and polymerase POLG causes distinct replication stalling phenotypes. Nucleic Acids Res 35:3238–3251CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    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 13:3219–3227CrossRefPubMedGoogle Scholar
  53. 53.
    Bourdon A, Minai L, Serre V, Jais JP, Sarzi E, Aubert S, Chretien D, de Lonlay P, Paquis-Flucklinger V, Arakawa H, Nakamura Y, Munnich A, Rotig A (2007) Mutation of RRM2B, encoding p53-controlled ribonucleotide reductase (p53R2), causes severe mitochondrial DNA depletion. Nat Genet 39:776–780CrossRefPubMedGoogle Scholar
  54. 54.
    Nishino I, Spinazzola A, Hirano M (1999) Thymidine phosphorylase gene mutations in MNGIE, a human mitochondrial disorder. Science 283:689–692CrossRefPubMedGoogle Scholar
  55. 55.
    Saada A, Shaag A, Mandel H, Nevo Y, Eriksson S, Elpeleg O (2001) Mutant mitochondrial thymidine kinase in mitochondrial DNA depletion myopathy. Nat Genet 29:342–344CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Ian J. Holt
    • 1
    Email author
  • Lawrence Kazak
    • 2
  • Aurelio Reyes
    • 2
  • Stuart R. Wood
    • 2
  1. 1.MRC-National Institute for Medical ResearchLondonUK
  2. 2.MRC-Mitochondrial Biology UnitCambridgeUK

Personalised recommendations