Direct visualization of RecBCD movement reveals cotranslocation of the RecD motor after chi recognition.
In Escherichia coli, chi (5'-GCTGGTGG-3') is a recombination hotspot recognized by the RecBCD enzyme. Recognition of chi 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 chi interaction has been "ejection" of the RecD motor from the holoenzyme at chi. 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 (approximately 0.6 microm/s). Those enzymes translocated to chi, paused, and continued at reduced velocity, without loss of RecD. We conclude that chi interaction induces a conformational change, resulting from binding of chi to RecC, and not from RecD ejection. This change is responsible for alteration of RecBCD function that persists for the duration of DNA translocation.
Handa
Naofumi
N
Section of Microbiology, University of California, Davis, Davis, California 95616, USA.
Bianco
Piero R
PR
Baskin
Ronald J
RJ
Kowalczykowski
Stephen C
SC
D019921:Biotinylation; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D043211:Exodeoxyribonuclease V / Q000378:metabolism*; D046911:Macromolecular Substances / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods*; D008957:Models, Genetic; D049329:Nanostructures; D010957:Plasmids / Q000378:metabolism; D021381:Protein Transport; D011995:Recombination, Genetic; D013997:Time Factors
D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D004247:DNA; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli