Advertisement

DNA Supercoiling Measurement in Bacteria

  • Yingting Liu
  • Zhi-Chun Hua
  • Fenfei Leng
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1703)

Abstract

DNA supercoiling plays critical roles in several essential DNA metabolic pathways, such as replication, transcription and recombination. Typically plasmid DNA molecules are used to measure DNA supercoiling status inside bacterial cells. In this chapter, we describe how to isolate plasmid DNA molecules from E. coli cells and determine DNA supercoiling density by 1% agarose gel electrophoresis containing chloroquine using plasmid pACYC184 as an example.

Key words

Agarose gel electrophoresis DNA supercoiling DNA topoisomers Supercoiling density 

References

  1. 1.
    Champoux JJ (2001) DNA topoisomerases: structure, function, and mechanism. Annu Rev Biochem 70:369–413CrossRefPubMedGoogle Scholar
  2. 2.
    Snoep JL, van der Weijden CC, Andersen HW, Westerhoff HV, Jensen PR (2002) DNA supercoiling in Escherichia coli is under tight and subtle homeostatic control, involving gene-expression and metabolic regulation of both topoisomerase I and DNA gyrase. Eur J Biochem 269:1662–1669CrossRefPubMedGoogle Scholar
  3. 3.
    Wang JC (1996) DNA topoisomerases. Annu Rev Biochem 65:635–692CrossRefPubMedGoogle Scholar
  4. 4.
    Zechiedrich EL, Khodursky AB, Bachellier S, Schneider R, Chen D, Lilley DM, Cozzarelli NR (2000) Roles of topoisomerases in maintaining steady-state DNA supercoiling in Escherichia coli. J Biol Chem 275:8103–8113CrossRefPubMedGoogle Scholar
  5. 5.
    White JH, Bauer WR (1986) Calculation of the twist and the writhe for representative models of DNA. J Mol Biol 189:329–341CrossRefPubMedGoogle Scholar
  6. 6.
    Cozzarelli NR, Boles TC, White JH (1990) A primer on the topology and geometry of DNA supercoiling. In: Cozzarelli NR, Wang JC (eds) DNA topology and its biological effects. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp 139–184Google Scholar
  7. 7.
    Bates AD, Maxwell A (2005) DNA topology, 2nd edn. Oxford University Press, OxfordGoogle Scholar
  8. 8.
    White JH, Cozzarelli NR, Bauer WR (1988) Helical repeat and linking number of surface-wrapped DNA. Science 241:323–327CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.Biomolecular Sciences InstituteFlorida International UniversityMiamiUSA
  2. 2.Department of Chemistry & BiochemistryFlorida International UniversityMiamiUSA
  3. 3.School of Life SciencesNanjing UniversityNanjingChina
  4. 4.Changzhou High-Tech Research Institute of Nanjing University and Jiangsu TargetPharma Laboratories Inc.ChangzhouChina

Personalised recommendations