Summary
Recent advances in optical imaging and molecular manipulation techniques have made it possible to observe the activity of individual enzymes and study the dynamic properties of processes that are challenging to elucidate using ensemble-averaging techniques. The use of single-molecule approaches has proven to be particularly successful in the study of the dynamic interactions between the components at the replication fork. In this section, we describe the methods necessary for in vitro single-molecule studies of prokaryotic replication systems. Through these experiments, accurate information can be obtained on the rates and processivities of DNA unwinding and polymerization. The ability to monitor in real time the progress of a single replication fork allows for the detection of short-lived, intermediate states that would be difficult to visualize in bulk-phase assays.
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References
Benkovic SJ, Valentine AM, Salinas F. Replisome-mediated DNA replication. Annu Rev Biochem 2001; 70:181–208
Lee JB, Hite RK, Hamdan SM, Xie XS, Richardson CC, van Oijen AM, . DNA primase acts as a molecular brake in DNA replication. Nature 2006; 439: 621–624
Hamdan SM, Johnson DE, Tanner NA, et al. Dynamic DNA helicase-DNA poly-merase interactions assure processive replication fork movement. Mol Cell 2007; 27:539–549
Tanner NA, Hamdan SM, Jergic S, Schaeffer PM, Dixon NE, van Oijen AM. Single-molecule studies of fork dynamics in Escherichia coli DNA replication. Nat Struct Mol Biol 2008; 15:170–176
Sofia SJ, Premnath VV, Merrill EW. Poly(ethylene oxide) grafted to silicon surfaces: grafting density and protein adsorption. Macromolecules 1998; 31:5059–6270
Acknowledgments
The authors would like to thank Charles Richardson and Nick Dixon for their generous gifts of T7 and E. coli replication proteins, respectively. The authors would also like to acknowledge contributions from Paul Blainey, Candice Etson, Jong-Bong Lee, and Joseph Loparo toward the development of the single-molecule replication assay.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Tanner, N.A., van Oijen, A.M. (2009). Single-Molecule Observation of Prokaryotic DNA Replication. In: Vengrova, S., Dalgaard, J. (eds) DNA Replication. Methods in Molecular Biology, vol 521. Humana Press. https://doi.org/10.1007/978-1-60327-815-7_22
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DOI: https://doi.org/10.1007/978-1-60327-815-7_22
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