In Escherichia coli, χ (5′-GCTGGTGG-3′) is a recombination hotspot recognized by the RecBCD enzyme. Recognition of χ reduces both nuclease activity and translocation speed of RecBCD and activates RecA-loading ability. RecBCD has two motor subunits, RecB and RecD, which act simultaneously but independently. A longstanding hypothesis to explain the changes elicited by χ interaction has been “ejection” of the RecD motor from the holoenzyme at χ. To test this proposal, we visualized individual RecBCD molecules labeled via RecD with a fluorescent nanoparticle. We could directly see these labeled, single molecules of RecBCD moving at up to 1835 bp/s (∼0.6 μm/s). Those enzymes translocated to χ, paused, and continued at reduced velocity, without loss of RecD. We conclude that χ interaction induces a conformational change, resulting from binding of χ to RecC, and not from RecD ejection. This change is responsible for alteration of RecBCD function that persists for the duration of DNA translocation.
Present address: University of Tokyo, Department of Medical Genome Sciences, Graduate School of Frontier Science & Institute of Medical Science, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan.
Present address: Center for Single Molecule Biophysics, Department of Microbiology and Biochemistry, State University of New York at Buffalo, Buffalo, New York 14214.
