Abstract
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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 745-750 |
| Number of pages | 6 |
| Journal | Molecular Cell |
| Volume | 17 |
| Issue number | 5 |
| DOIs | |
| State | Published - Mar 4 2005 |
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- Molecular Biology
Cite this
Direct visualization of RecBCD movement reveals cotranslocation of the RecD motor after χ recognition. / Handa, Naofumi; Bianco, Piero R.; Baskin, Ronald J.; Kowalczykowski, Stephen C.
In: Molecular Cell, Vol. 17, No. 5, 04.03.2005, p. 745-750.Research output: Contribution to journal › Article
}
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.
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.
UR - http://www.scopus.com/inward/record.url?scp=14644412914&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=14644412914&partnerID=8YFLogxK
U2 - 10.1016/j.molcel.2005.02.011
DO - 10.1016/j.molcel.2005.02.011
M3 - Article
C2 - 15749023
AN - SCOPUS:14644412914
VL - 17
SP - 745
EP - 750
JO - Molecular Cell
JF - Molecular Cell
SN - 1097-2765
IS - 5
ER -