Processive translocation and DNA unwinding by individual RecBCD enzyme molecules

RecBCD enzyme is a processive DNA helicase(1) and nuclease(2) that particip ates in the repair of chromosomal DNA through homologous recombination(3,4) . We have visualized directly the movement of individual RecBCD enzymes on single molecules of double-stranded DNA (dsDNA). Detection involves the opt ical trapping of solitary, fluorescently tagged dsDNA molecules that are at tached to polystyrene beads, and their visualization by fluorescence micros copy(5,6). Both helicase translocation and DNA unwinding are monitored by t he displacement of fluorescent dye from the DNA by the enzyme(7). Here we s how that unwinding is both continuous and processive, occurring at a maximu m rate of 972 +/- 172 base pairs per second (0.30 mum s(-1)), with as many as 42,300 base pairs of dsDNA unwound by a single RecBCD enzyme molecule. T he mean behaviour of the individual RecBCD enzyme molecules corresponds to that observed in bulk solution.