Processive translocation and DNA unwinding by individual RecBCD enzyme molecules

Abstract

RecBCD enzyme is a processive DNA helicase and nuclease that participates in the repair of chromosomal DNA through homologous recombination. We have visualized directly the movement of individual RecBCD enzymes on single molecules of double-stranded DNA (dsDNA). Detection involves the optical trapping of solitary, fluorescently tagged dsDNA molecules that are attached to polystyrene beads, and their visualization by fluorescence microscopy. Both helicase translocation and DNA unwinding are monitored by the displacement of fluorescent dye from the DNA by the enzyme. Here we show that unwinding is both continuous and processive, occurring at a maximum rate of 972 +/- 172 base pairs per second (0.30 microm s(-1)), with as many as 42,300 base pairs of dsDNA unwound by a single RecBCD enzyme molecule. The mean behaviour of the individual RecBCD enzyme molecules corresponds to that observed in bulk solution.

Simultaneous separation and concentration of micro- and nano-particles by optically induced electrokinetics

“…In this paper, we discuss the application of optically induced dielectrophoresis (ODEP) to rapidly separate and assemble micro-/nanometer-sized polystyrene beads, which is very important in developing advanced bio-sensing devices. For example, DNAs [5], cells [6], or proteins [7], can all be immobilized or hybridized onto the surface of polystyrene beads, and then be easily separated, or assembled for later biomedical or engineering applications by manipulating the larger polystyrene beads.…”

Erratum to: Structure and Mechanisms of SF1 DNA Helicases

“…These techniques are particularly useful for visualizing kinetic events that are "hidden" within ensemble experiments. Recognition of the Chi sequence in DNA causes RecBCD to pause and reduce its translocation rate to approximately one-half the initial rate [139][140][141] resulting in a switch in motor usage with RecD being the lead motor prior to Chi and RecB after the Chi sites [142]. Magnetic tweezer analysis reported the average unwinding rate by RecBCD to be 900 bp/s [143] and the processivity of the complex to be ~1.…”

“…RecB helicase is activated through an interaction with RecC. RecB is a poor helicase by itself [186], but in complex with RecC is highly processive [139,182,183,187]. Interaction with the accessory protein RecC is suggested to relieve an inhibitory function of the 2B subdomain of RecB [188].…”

The importance of surfaces in single-molecule bioscience

“…[68][69][70][71] These experiments utilize fluorescently tagged protein or DNA molecules and rely on a laminar flow system, which has input channels for multiple buffers that do not mix as they pass through the sample chamber. The experiments are conducted using the optical trap to capture a DNA molecule tethered to a polystyrene bead through a biotin-streptavidin interaction.…”