Skip to main content

Analysis of Branched DNA Replication and Recombination Intermediates from Prokaryotic Cells by Two-Dimensional (2D) Native–Native Agarose Gel Electrophoresis

  • Protocol
  • First Online:

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

Abstract

Branched DNA molecules are generated by the essential processes of replication and recombination. Owing to their distinctive extended shapes, these intermediates migrate differently from linear double-stranded DNA under certain electrophoretic conditions. However, these branched species exist in the cell at much low abundance than the bulk linear DNA. Consequently, branched molecules cannot be visualized by conventional electrophoresis and ethidium bromide staining. Two-dimensional native–native agarose electrophoresis has therefore been developed as a method to facilitate the separation and visualization of branched replication and recombination intermediates. A wide variety of studies have employed this technique to examine branched molecules in eukaryotic, archaeal, and bacterial cells, providing valuable insights into how DNA is duplicated and repaired in all three domains of life.

This is a preview of subscription content, log in via an institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   119.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. 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 

  2. 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 

  3. Robinson NP, Dionne I, Lundgren M, Marsh VL, Bernander R, Bell SD (2004) Identification of two origins of replication in the single chromosome of the archaeon Sulfolobus solfataricus. Cell 116:25–38

    Article  PubMed  CAS  Google Scholar 

  4. Robinson NP, Blood KA, McCallum SA, Edwards PA, Bell SD (2007) Sister chromatid junctions in the hyperthermophilic archaeon Sulfolobus solfataricus. EMBO J 26:816–824

    Article  PubMed  CAS  Google Scholar 

  5. Robinson NP, Bell SD (2007) Extrachromosomal element capture and the evolution of multiple replication origins in archaeal chromosomes. Proc Natl Acad Sci U S A 104:5806–5811

    Article  PubMed  CAS  Google Scholar 

  6. Matsunaga F, Forterre P, Ishino Y, Myllykallio H (2001) In vivo interactions of archaeal Cdc6/Orc1 and minichromosome maintenance proteins with the replication origin. Proc Natl Acad Sci U S A 98:11152–11157

    Article  PubMed  CAS  Google Scholar 

  7. Duggin IG, Bell SD (2009) Termination structures in the Escherichia coli chromosome replication fork trap. J Mol Biol 387:532–539

    Article  PubMed  CAS  Google Scholar 

  8. Sambrook J, Russell DW (2001) Molecular cloning. A laboratory manual, 3rd ed, Cold Spring Harbor Laboratory Press, New York.

    Google Scholar 

  9. Friedman KL, Brewer BJ (1995) Analysis of replication intermediates by two-dimensional agarose gel electrophoresis. Methods Enzymol 262:613–627

    Article  PubMed  CAS  Google Scholar 

  10. Lopes M, Cotta-Ramusino C, Liberi G, Foiani M (2003) Branch migrating sister chromatid junctions form at replication origins through Rad51/Rad52-independent mechanisms. Mol Cell 12:1499–1510

    Article  PubMed  CAS  Google Scholar 

  11. Lucas I, Hyrien O (2000) Hemicatenanes form upon inhibition of DNA replication. Nucleic Acids Res 28:2187–2193

    Article  PubMed  CAS  Google Scholar 

  12. Oh SD, Jessop L, Lao JP, Allers T, Lichten M, Hunter N (2009) Stabilization and electrophoretic analysis of meiotic recombination intermediates in Saccharomyces cerevisiae. Methods Mol Biol 557:209–234

    Article  PubMed  CAS  Google Scholar 

  13. Liberi G, Maffioletti G, Lucca C, Chiolo I, Baryshnikova A, Cotta-Ramusino C, Lopes M, Pellicioli A, Haber JE, Foiani M (2005) Rad51-dependent DNA structures accumulate at damaged replication forks in sgs1 mutants defective in the yeast ortholog of BLM RecQ helicase. Genes Dev 19:339–350

    Article  PubMed  CAS  Google Scholar 

  14. Duggin IG, McCallum SA, Bell SD (2008) Chromosome replication dynamics in the archaeon Sulfolobus acidocaldarius. Proc Natl Acad Sci U S A 105:16737–16742

    Article  PubMed  CAS  Google Scholar 

  15. Banfalvi G (2011) Overview of cell synchronization. Methods Mol Biol 761:1–23

    Google Scholar 

  16. Bates D, Epstein J, Boye E, Fahrner K, Berg H, Kleckner N (2005) The Escherichia coli baby cell column: a novel cell synchronization method provides new insight into the bacterial cell cycle. Mol Microbiol 57:380–391

    Article  PubMed  CAS  Google Scholar 

  17. Ferullo DJ, Cooper DL, Moore HR, Lovett ST (2009) Cell cycle synchronization of Escherichia coli using the stringent response, with fluorescence labeling assays for DNA content and replication. Methods 48:8–13

    Article  PubMed  CAS  Google Scholar 

  18. Helmstetter CE, Thornton M, Romero A, Eward KL (2003) Synchrony in human, mouse and bacterial cell cultures – a comparison. Cell Cycle 2:42–45

    Article  PubMed  CAS  Google Scholar 

  19. Mesner LD, Dijkwel PA, Hamlin JL (2009) Purification of restriction fragments containing replication intermediates from complex genomes for 2-D gel analysis. Methods Mol Biol 521:121–137

    Article  PubMed  CAS  Google Scholar 

  20. Krysan PJ, Calos MP (1991) Replication initiates at multiple locations on an autonomously replicating plasmid in human cells. Mol Cell Biol 11:1464–1472

    PubMed  CAS  Google Scholar 

  21. 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 25:5358–5371

    Article  PubMed  CAS  Google Scholar 

  22. Hyrien O, Mechali M (1992) Plasmid replication in Xenopus eggs and egg extracts: a 2D gel electrophoretic analysis. Nucleic Acids Res 20:1463–1469

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

N.P.R. would like to thank Johanna Syrjanen (Society for General Microbiology Vacation student, 2010) for performing the 2D gels used as examples in Figs. 1 and 2.

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media, New York

About this protocol

Cite this protocol

Robinson, N.P. (2013). Analysis of Branched DNA Replication and Recombination Intermediates from Prokaryotic Cells by Two-Dimensional (2D) Native–Native 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_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-565-1_3

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-564-4

  • Online ISBN: 978-1-62703-565-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics