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Single-Molecule Observation of Prokaryotic DNA Replication

  • Hylkje J. Geertsema
  • Karl E. Duderstadt
  • Antoine M. van Oijen
Part of the Methods in Molecular Biology book series (MIMB, volume 1300)

Abstract

Replication of DNA requires the coordinated activity of a number of proteins within a multiprotein complex, the replisome. Recent advances in single-molecule techniques have enabled the observation of dynamic behavior of individual replisome components and of the replisome as a whole, aspects that previously often have been obscured by ensemble averaging in more classical solution-phase biochemical experiments. To improve robustness and reproducibility of single-molecule assays of replication and allow objective analysis and comparison of results obtained from such assays, common practices should be established. Here, we describe the technical details of two assays to study replisome activity. In one, the kinetics of replication are observed as length changes in DNA molecules mechanically stretched by a laminar flow applied to attached beads. In the other, fluorescence imaging is used to determine both the kinetics and stoichiometry of individual replisome components. These in vitro single-molecule methods allow for elucidation of the dynamic behavior of individual replication proteins of prokaryotic replication systems.

Key words

DNA replication Single molecule T7 bacteriophage E. coli 

Notes

Acknowledgements

The authors would like to thank Charles Richardson and Nick Dixon for their generous gifts of T7 and E. coli replication proteins, respectively. AMvO would like to acknowledge funding from the Netherlands Organization for Scientific Research (NWO; Vici 680-47-607) and the European Research Council (ERC Starting 281098). KED acknowledges postdoctoral funding from the Human Frontier Science Program.

References

  1. 1.
    Benkovic SJ, Valentine AM, Salinas F (2001) Replisome-mediated DNA replication. Annu Rev Biochem 70:181–208CrossRefPubMedGoogle Scholar
  2. 2.
    Lee JB, Hite RK, Hamdan SM, Xie XS, Richardson CC, van Oijen AM (2006) DNA primase acts as a molecular brake in DNA replication. Nature 439:621–624CrossRefPubMedGoogle Scholar
  3. 3.
    Hamdan SM, Loparo JJ, Takahashi M, Richardson CC, van Oijen AM (2009) Dynamics of individual DNA replication loops reveal temporal control of lagging-strand synthesis. Nature 457:336–339CrossRefPubMedCentralPubMedGoogle Scholar
  4. 4.
    Tanner NA, Hamdan SM, Jergic S, Loscha KV, Schaeffer PM, Dixon NE, van Oijen AM (2008) Single-molecule studies of fork dynamics in Escheria coli DNA replication. Nat Struct Mol Biol 15:170–175CrossRefPubMedGoogle Scholar
  5. 5.
    Loparo JJ, Kulczyk AW, Richardson CC, van Oijen AM (2011) Simultaneous single-molecule measurements of phage T7 replisome composition and function reveal the mechanism of polymerase exchange. Proc Natl Acad Sci U S A 108:3584–3589CrossRefPubMedCentralPubMedGoogle Scholar
  6. 6.
    Geertsema HJ, Kulczyk AW, Richarson CC, van Oijen AM (2014) Single-molecule studies of polymerase dynamics and stoichiometry at the bacteriophage T7 replication machinery. Proc Natl Acad Sci U S A 111:4073–4078CrossRefPubMedCentralPubMedGoogle Scholar
  7. 7.
    Georgescu RE, Kurth I, O’Donnell ME (2012) Single-molecule studies reveal the function of a third polymerase in the replisome. Nat Struct Mol Biol 19:113–116CrossRefGoogle Scholar
  8. 8.
    Yardimci H, Loveland AB, Habuchi S, van Oijen AM, Walter JC (2010) Uncoupling of sister replisomes during Eukaryotic DNA replication. Mol Cell 40:834–840CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Hylkje J. Geertsema
    • 1
  • Karl E. Duderstadt
    • 1
  • Antoine M. van Oijen
    • 1
    • 2
    • 3
  1. 1.University of GroningenGroningenThe Netherlands
  2. 2.Centre for Synthetic BiologyUniversity of GroningenGroningenThe Netherlands
  3. 3.Zernike Institute for Advanced MaterialsUniversity of GroningenGroningenThe Netherlands

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