Direct visualization of RecBCD movement reveals cotranslocation of the RecD motor after Χ recognition

In Escherichia coli, X (5'-GCTGGTGG-3') is a recombination hotspot recognized by the RecBCD enzyme. Recognition of X reduces both nuclease activity and translocation speed of RecBCD and activates RecAloading ability. RecBCD has two motor subunits, RecB and RecD, which act simultaneously but independently. A longstanding hypothesis to explain the changes elicited by X interaction has been"ejection" of the RecD motor from the holoenzyme at X. To test this proposal,we visualized individual RecBCD molecules labeled via RecD with a fluorescentnanoparticle. We could directly see these labeled, single molecules of RecBCDmoving at up to 1835 bp/s (0.6 lim/s). Those enzymes translocated to X, paused, and continued at reduced velocity, without loss of RecD. We conclude that Xinteraction induces a conformational change, resulting from binding of X to RecC, and not from RecD ejection. This change is responsible for alteration of RecBCD function that persists for the duration of DNA translocation.