Processive translocation and DNA unwinding by individual RecBCD enzyme molecules

RecBCD enzyme is a processive DNA helicase(1) and nuclease(2) that participates 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 optical trapping of solitary, fluorescently tagged dsDNA molecules that are attached to polystyrene beads, and their visualization by fluorescence microscopy(5,6). Both helicase translocation and DNA unwinding are monitored by the displacement of fluorescent dye from the DNA by the enzyme(7). Here we show that unwinding is both continuous and processive, occurring at a maximum 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. The mean behaviour of the individual RecBCD enzyme molecules corresponds to that observed in bulk solution

RecBCD, SbcCD and ExoI Process a Substrate Created by Convergent Replisomes to Complete DNA Replication

The accurate completion of DNA replication on the chromosome requires RecBCD and structure specific SbcCD and ExoI nucleases. However, the substrates and mechanism by which this reaction occurs remains unknown. Here we show that these completion enzymes operate on plasmid substrates containing two replisomes, but are not required for plasmids containing one replisome. Completion on the two‐replisome plasmids requires RecBCD, but does not require RecA and no broken intermediates accumulate in its absence, indicating that the completion reaction occurs normally in the absence of any double‐strand breaks. Further, similar to the chromosome, we show that when the normal completion reaction is prevented, an aberrant RecA‐mediated recombination process leads to amplifications that drive most of the instabilities associated with the two‐replisome substrates. The observations imply that the substrate SbcCD, ExoI and RecBCD act upon in vivo is created specifically by two convergent replisomes, and demonstrate that the function of RecBCD in completing replication is independent of double‐strand break repair, and likely promotes joining of the strands of the convergent replication forks