Single-molecule studies of RecBCD.
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.
Perkins
Thomas T
TT
MCD Biology, JILA, NIST & CU, University of Colorado at Boulder, Boulder, CO, USA.
Li
Hung-Wen
HW
D001710:Biotin / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D043211:Exodeoxyribonuclease V / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D052898:Optical Tweezers
D029968:Escherichia coli Proteins; D001710:Biotin; D004247:DNA; D043211:Exodeoxyribonuclease V