Abstract
RecBCD is a processive molecular motor composed of two independent helicase domains and a nuclease domain. Understanding the molecular mechanism of its motor activity involves, in part, determining RecBCD’s translocation properties (e.g., velocity, propensity to pause, pause duration). Single-molecule techniques, in general, and optical trapping, specifically, provide for measuring the translocation of individual molecules along DNA. We developed a high-spatial resolution optical-trapping assay for RecBCD. The RecBCD is anchored to the surface via a genetically engineered biotin. This RecBCD-bio exhibited native activity, as measured by biochemical assays. Motion is continuous down to a detection limit of 2 nm, implying a unitary step size below 6 base pairs. Unexpectedly, the catalytic rate changes abruptly and persists at different values for tens of seconds. This technically demanding, high-resolution optical-trapping assay is complemented by a simpler single-molecule assay—the tethered particle motion assay.
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Acknowledgments
The authors would like to thank Ashley Carter for critical reading of the manuscript, and Ashley Carter and D. Hern Paik for figure preparation. This work was supported by a Burroughs Wellcome Fund Career Award in the Biomedical Sciences (TTP), the National Science Foundation (NSF Phys-0404286 to TTP), National Research Council (Taiwan; to H.-W.L.), and National Institute of Standards and Technology (NIST). Mention of commercial products is for information only; it does not imply NIST recommendation or endorsement, nor does it imply that the products mentioned are necessarily the best available for the purpose. TTP is a staff member of NIST’s Quantum Physics Division.
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Perkins, T.T., Li, HW. (2009). Single-Molecule Studies of RecBCD. In: Abdelhaleem, M. (eds) Helicases. Methods in Molecular Biology, vol 587. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-355-8_11
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DOI: https://doi.org/10.1007/978-1-60327-355-8_11
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