TY - JOUR
T1 - Direct visualization of RecBCD movement reveals cotranslocation of the RecD motor after χ recognition
AU - Handa, Naofumi
AU - Bianco, Piero R.
AU - Baskin, Ronald J.
AU - Kowalczykowski, Stephen C.
N1 - Funding Information:
We would like to thank I. Amitani, M. Dillingham, K. Morimatsu, Z. Ozsoy, J. Siino, M. Spies for valuable discussion and the members of the Kowalczykowski laboratory for their critical reading of the manuscript. This work was supported by the Japan Society for the Promotion of Science Postdoctoral Fellowship for Research Abroad to N.H., and by National Institutes of Health grants GM-41347 and GM-64745.
PY - 2005/3/4
Y1 - 2005/3/4
N2 - 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.
AB - 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.
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U2 - 10.1016/j.molcel.2005.02.011
DO - 10.1016/j.molcel.2005.02.011
M3 - Article
C2 - 15749023
AN - SCOPUS:14644412914
SN - 1097-2765
VL - 17
SP - 745
EP - 750
JO - Molecular Cell
JF - Molecular Cell
IS - 5
ER -