FIG. 7.

Single-molecule analysis of the RecBCD enzyme. (A) Direct visualization of DNA translocation using fluorescently labeled DNA. A single lambda phage DNA molecule is attached to a polystyrene bead, and YOYO-1 (a fluorescent DNA binding dye) is then bound. The bead is held in an optical trap, with the DNA molecule stretched out behind it by solution flow. A single molecule of the RecBCD enzyme is able to bind to the free DNA end. Upon the addition of ATP, the RecBCD enzyme unwinds and degrades the duplex. This is observed as a progressive shortening of the fluorescent DNA in an epifluorescence microscope. (A movie of this experiment can be viewed at http://microbiology.ucdavis.edu/sklab/kowalczykowskilab.htm.) The cartoon is not to scale; the stretched lambda DNA is 48.5 kbp (∼15 to 16 μM) long and binds several thousand molecules of YOYO-1 dye. (B) Direct monitoring of RecBCD translocation by tracking of a fluorescent nanoparticle (40-nm diameter). The instrument setup is as described above for A except that the DNA is not labeled. Instead of monitoring DNA degradation, the fluorescent nanoparticle is attached to the translocating enzyme via a biotin moiety on the RecD subunit. The position of the nanoparticle relative to the position of the optical laser trap measures DNA translocation. (C) RecBCD enzyme translocation on DNA monitored by tethered-particle light microscopy. The biotinylated RecBCD enzyme is bound to a streptavidin-coated bead. The RecBCD enzyme then binds to the free end of a surface-attached DNA molecule (∼1.4 kbp), effectively tethering the bead to the surface with the DNA as a linker. The translocation of RecBCD along the DNA pulls the bead toward the surface, which places an increasing constraint on the bead's Brownian motion that can be monitored by light microscopy. (D) RecBCD enzyme translocation on DNA monitored by high-resolution optical trapping. A biotinylated RecBCD enzyme is attached to a streptavidin-coated surface (alternatively, but not shown, a second optical trap can be used). RecBCD, attached to a bead, can capture the free end of a 7-kbp DNA molecule. The bead is held in an optical trap with a force clamp. Upon DNA translocation, the RecBCD enzyme pulls on the DNA and generates force against the trapped bead. A feedback mechanism moves the surface stage toward the trapped bead to maintain a constant force. The instrument can assess the effects of applied force against translocation and measure the movement of the enzyme with nanometer resolution.