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.
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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.
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Geertsema, H.J., Duderstadt, K.E., van Oijen, A.M. (2015). Single-Molecule Observation of Prokaryotic DNA Replication. In: Vengrova, S., Dalgaard, J. (eds) DNA Replication. Methods in Molecular Biology, vol 1300. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2596-4_14
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DOI: https://doi.org/10.1007/978-1-4939-2596-4_14
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2595-7
Online ISBN: 978-1-4939-2596-4
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