Abstract
Over the recent years single-molecule fluorescence resonance energy transfer (smFRET) technique has proven to be one of the most powerful tools for revealing mechanistic insights into helicase activities. Here we describe details of single-molecule FRET assays for probing DNA unwinding activities as well as functional dynamics by replicative helicases in real time. The ability of smFRET to measure the behavior of biomolecules at a nanometer scale enabled us to address how the leading and lagging strand synthesis are coordinated during DNA replication, to resolve DNA unwinding steps of Bacteriophage T7 helicase, and to observe heterogeneous unwinding patterns modulated by the DNA binding domain of E1 helicase. These single-molecule FRET assays are generally applicable to other replicative and nonreplicative hexameric helicases.
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Acknowledgments
The authors would like to thank all the members of Ha laboratory for valuable discussions and experimental help. These studies were supported by grants from the National Institute of Health and the National Science Foundation. T.H. is an Investigator with the Howard Hughes Medical Institute.
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Lee, SJ., Syed, S., Ha, T. (2018). Single-Molecule FRET Analysis of Replicative Helicases. In: Lavelle, C. (eds) Molecular Motors. Methods in Molecular Biology, vol 1805. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8556-2_12
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DOI: https://doi.org/10.1007/978-1-4939-8556-2_12
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