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Plasmid DNA Topology Assayed by Two-Dimensional Agarose Gel Electrophoresis

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DNA Electrophoresis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1054))

Abstract

Two-dimensional (2D) agarose gel electrophoresis is nowadays one of the best methods available to analyze DNA molecules with different masses and shapes. The possibility to use nicking enzymes and intercalating agents to change the twist of DNA during only one or in both runs, improves the capacity of 2D gels to discern molecules that apparently may look alike. Here we present protocols where 2D gels are used to understand the structure of DNA molecules and its dynamics in living cells. This knowledge is essential to comprehend how DNA topology affects and is affected by all the essential functions that DNA is involved in: replication, transcription, repair and recombination.

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References

  1. Schvartzman JB, Stasiak A (2004) A topological view of the replicon. EMBO Rep 5:256–261

    Article  PubMed  CAS  Google Scholar 

  2. Watson JD, Crick FHC (1953) Genetical implications of the structure of deoxyribonucleic acids. Nature 171:964–967

    Article  PubMed  CAS  Google Scholar 

  3. Watson JD, Crick FHC (1953) Molecular structure of nucleic acids. Nature 161:737–738

    Article  Google Scholar 

  4. Cairns J (1963) The bacterial chromosome and its manner of replication as seen by autoradiography. J Mol Biol 6:208–213

    Article  PubMed  CAS  Google Scholar 

  5. Wang JC (1969) Degree of superhelicity of covalently closed cyclic DNA’s from Escherichia coli. J Mol Biol 43:263–272

    Article  PubMed  CAS  Google Scholar 

  6. Wang JC (1971) Interaction between DNA and an Escherichia coli protein omega. J Mol Biol 55:523–533

    Article  PubMed  CAS  Google Scholar 

  7. Weil R, Vinograd J (1963) The cyclic helix and cyclic coil forms of polyoma viral DNA. Proc Natl Acad Sci U S A 50:730–739

    Article  PubMed  CAS  Google Scholar 

  8. Dulbecco R, Vogt M (1963) Evidence for a ring structure of polyoma virus DNA. Proc Natl Acad Sci U S A 50:236–243

    Article  PubMed  CAS  Google Scholar 

  9. Thorne HV (1966) Electrophoretic separation of polyoma virus DNA from host cell DNA. Virology 29:234–239

    Article  PubMed  CAS  Google Scholar 

  10. Stellwagen NC (2009) Electrophoresis of DNA in agarose gels, polyacrylamide gels and in free solution. Electrophoresis 30(Suppl 1):S188–S195

    Article  PubMed  Google Scholar 

  11. Bauer W, Vinograd J (1968) The interaction of closed circular DNA with intercalative dyes. J Mol Biol 33:141–171

    Article  PubMed  CAS  Google Scholar 

  12. Bauer W, Vinograd J (1970) Interaction of closed circular DNA with intercalative dyes. II. The free energy of superhelix formation in SV40 DNA. J Mol Biol 47:419–435

    Article  PubMed  CAS  Google Scholar 

  13. Bauer W, Vinogradj (1970) The interaction of closed circular DNA with intercalative dyes. 3. Dependence of the buoyant density upon superhelix density and base composition. J Mol Biol 54:281–298

    Google Scholar 

  14. Keller W (1975) Determination of the number of superhelical turns in simian virus 40 DNA by gel electrophoresis. Proc Natl Acad Sci U S A 72:4876–4880

    Article  PubMed  CAS  Google Scholar 

  15. LePecq JB, Paoletti C (1967) A fluorescent complex between ethidium bromide and nucleic acids. Physical–chemical characterization. J Mol Biol 27:87–106

    Article  PubMed  CAS  Google Scholar 

  16. Lerman LS (1961) Structural considerations in the interaction of DNA and acridines. J Mol Biol 3:18–30

    Article  PubMed  CAS  Google Scholar 

  17. Pulleyblank DE, Morgan AR (1975) The sense of naturally occurring superhelices and the unwinding angle of intercalated ethidium. J Mol Biol 91:1–13

    Article  PubMed  CAS  Google Scholar 

  18. Bell L, Byers B (1983) Separation of branched from linear DNA by two-dimensional gel electrophoresis. Anal Biochem 130:527–535

    Article  PubMed  CAS  Google Scholar 

  19. Brewer BJ, Fangman WL (1987) The localization of replication origins on ARS plasmids in S. cerevisiae. Cell 51:463–471

    Article  PubMed  CAS  Google Scholar 

  20. Huberman JA, Spotila LD, Nawotka KA, El-Assouli SM, Davis LR (1987) The in vivo replication origin of the yeast 2 mm plasmid. Cell 51:473–481

    Article  PubMed  CAS  Google Scholar 

  21. Lee CH, Mizusawa H, Kakefuda T (1981) Unwinding of double-stranded DNA helix by dihydration. Proc Natl Acad Sci U S A 78:2838–2842

    Article  PubMed  CAS  Google Scholar 

  22. Minden JS, Marians KJ (1985) Replication of pBR322 DNA in vitro with purified proteins. J Biol Chem 260:9316–9325

    PubMed  CAS  Google Scholar 

  23. Oppenheim A (1981) Separation of closed circular DNA from linear DNA by electrophoresis in two dimensions in agarose gels. Nucleic Acids Res 9:6805–6812

    Article  PubMed  CAS  Google Scholar 

  24. Sundin O, Varshavsky A (1980) Terminal stages of SV40 DNA replication proceed via multiply intertwined catenated dimers. Cell 21:103–114

    Article  PubMed  CAS  Google Scholar 

  25. Sundin O, Varshavsky A (1981) Arrest of segregation leads to accumulation of highly intertwined catenated dimers dissection of the final stages of SV40 DNA replication. Cell 25:659–669

    Article  PubMed  CAS  Google Scholar 

  26. Ferrandiz MJ, Martin-Galiano AJ, Schvartzman JB, de la Campa AG (2010) The genome of Streptococcus pneumoniae is organized in topology-reacting gene clusters. Nucleic Acids Res 38:3570–3581

    Article  PubMed  CAS  Google Scholar 

  27. Mayan-Santos MD, Martinez-Robles ML, Hernandez P, Krimer D, Schvartzman JB (2007) DNA is more negatively supercoiled in bacterial plasmids than in minichromosomes isolated from budding yeast. Electrophoresis 28:3845–3853

    Article  PubMed  CAS  Google Scholar 

  28. Mirkin SM (2001) DNA topology: fundamentals, John Wiley & Sons, Inc. DOI: 10.1038/npg.els.0001038

  29. Lopez V, Martinez-Robles ML, Hernandez P, Krimer DB, Schvartzman JB (2012) Topo IV is the topoisomerase that knots and unknots sister duplexes during DNA replication. Nucleic Acids Res 40:3563–3573

    Article  PubMed  CAS  Google Scholar 

  30. Martinez-Robles ML et al (2009) Interplay of DNA supercoiling and catenation during the segregation of sister duplexes. Nucleic Acids Res 37:5126–5137

    Article  PubMed  CAS  Google Scholar 

  31. Adams DE, Shekhtman EM, Zechiedrich EL, Schmid MB, Cozzarelli NR (1992) The role of topoisomerase-IV in partitioning bacterial replicons and the structure of catenated intermediates in DNA replication. Cell 71:277–288

    Article  PubMed  CAS  Google Scholar 

  32. Olavarrieta L, Hernández P, Krimer DB, Schvartzman JB (2002) DNA knotting caused by head-on collision of transcription and replication. J Mol Biol 322:1–6

    Article  PubMed  CAS  Google Scholar 

  33. Olavarrieta L, Martínez-Robles ML, Hernández P, Krimer DB, Schvartzman JB (2002) Knotting dynamics during DNA replication. Mol Microbiol 46:699–707

    Article  PubMed  CAS  Google Scholar 

  34. Olavarrieta L et al (2002) Supercoiling, knotting and replication fork reversal in partially replicated plasmids. Nucleic Acids Res 30:656–666

    Article  PubMed  CAS  Google Scholar 

  35. Sogo JM et al (1999) Formation of knots in partially replicated DNA molecules. J Mol Biol 286:637–643

    Article  PubMed  CAS  Google Scholar 

  36. Viguera E et al (1996) The ColE1 unidirectional origin acts as a polar replication fork pausing site. J Biol Chem 271:22414–22421

    Article  PubMed  CAS  Google Scholar 

  37. Fierro-Fernandez M, Hernandez P, Krimer DB, Schvartzman JB (2007) Replication fork reversal occurs spontaneously after digestion but is constrained in supercoiled domains. J Biol Chem 282:18190–18196

    Article  PubMed  CAS  Google Scholar 

  38. Fierro-Fernandez M, Hernandez P, Krimer DB, Stasiak A, Schvartzman JB (2007) Topological locking restrains replication fork reversal. Proc Natl Acad Sci U S A 104:1500–1505

    Article  PubMed  CAS  Google Scholar 

  39. Long DT, Kreuzer KN (2008) Regression supports two mechanisms of fork processing in phage T4. Proc Natl Acad Sci U S A 105:6852–6857

    Article  PubMed  CAS  Google Scholar 

  40. Long DT, Kreuzer KN (2009) Fork regression is an active helicase-driven pathway in bacteriophage T4. EMBO Rep 10:394–399

    Article  PubMed  CAS  Google Scholar 

  41. Lopes M et al (2001) The DNA replication checkpoint response stabilizes stalled replication forks. Nature 412:557–561

    Article  PubMed  CAS  Google Scholar 

  42. Pohlhaus J, Kreuzer K (2006) Formation and processing of stalled replication forks—utility of two-dimensional agarose gels. Methods Enzymol 409:477–493

    Article  PubMed  CAS  Google Scholar 

  43. Postow L et al (2001) Positive torsional strain causes the formation of a four-way junction at replication forks. J Biol Chem 276:2790–2796

    Article  PubMed  CAS  Google Scholar 

  44. Sogo JM, Lopes M, Foiani M (2002) Fork reversal and ssDNA accumulation at stalled replication forks owing to checkpoint defects. Science 297:599–602

    Article  PubMed  CAS  Google Scholar 

  45. Viguera E, Hernandez P, Krimer DB, Lurz R, Schvartzman JB (2000) Visualisation of plasmid replication intermediates containing reversed forks. Nucleic Acids Res 28:498–503

    Article  PubMed  CAS  Google Scholar 

  46. Santamaría D, Hernández P, Martínez-Robles ML, Krimer DB, Schvartzman JB (2000) Premature termination of DNA replication in plasmids carrying two inversely oriented ColE1 origins. J Mol Biol 300:75–82

    Article  PubMed  Google Scholar 

  47. Vologodskii A (1998) Exploiting circular DNA. Proc Natl Acad Sci U S A 95:4092–4093

    Article  PubMed  CAS  Google Scholar 

  48. Wang JC, Peck LJ, Becherer K (1983) DNA supercoiling and its effects on DNA structure and function. Cold Spring Harb Symp Quant Biol 47(Pt 1):85–91

    Article  PubMed  Google Scholar 

  49. Horowitz DS, Wang JC (1984) Torsional rigidity of DNA and length dependence of the free energy of DNA supercoiling. J Mol Biol 173:75–91

    Article  PubMed  CAS  Google Scholar 

  50. Salceda J, Fernandez X, Roca J (2006) Topoisomerase II, not topoisomerase I, is the proficient relaxase of nucleosomal DNA. EMBO J 25:2575–2583

    Article  PubMed  CAS  Google Scholar 

  51. Friedman KL, Brewer BJ (1995) Analysis of replication intermediates by two-dimensional agarose gel electrophoresis. In: Campbell JL (ed) DNA replication, methods in enzymology, vol 262. Academic Press Inc., San Diego, CA, pp 613–627

    Chapter  Google Scholar 

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Acknowledgements

We acknowledge current and past members of the laboratory for their continuous suggestions and support. We would like to strengthen that this work could not be accomplished without the continuous support and constructive criticism of Andrzej Stasiak. This work was sustained by grant BFU2011-22489 to J.B.S. from the Spanish Ministerio de Economía y Competitividad.

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Authors and Affiliations

  1. Department of Cell and Molecular Biology, Centro de Investigaciones Biológicas (CSIC), Madrid, Spain

    Jorge B. Schvartzman, María-Luisa Martínez-Robles, Pablo Hernández & Dora B. Krimer

Authors
  1. Jorge B. Schvartzman
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  2. María-Luisa Martínez-Robles
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  3. Pablo Hernández
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  4. Dora B. Krimer
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Editors and Affiliations

  1. Institute of Cell Biology, University of Edinburgh, Edinburgh, United Kingdom

    Svetlana Makovets

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Schvartzman, J.B., Martínez-Robles, ML., Hernández, P., Krimer, D.B. (2013). Plasmid DNA Topology Assayed by Two-Dimensional Agarose Gel Electrophoresis. In: Makovets, S. (eds) DNA Electrophoresis. Methods in Molecular Biology, vol 1054. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-565-1_7

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  • DOI: https://doi.org/10.1007/978-1-62703-565-1_7

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-564-4

  • Online ISBN: 978-1-62703-565-1

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