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

Naofumi Handa, Piero R. Bianco, Ronald J. Baskin, Stephen C. Kowalczykowski

Research output: Contribution to journalArticle

88 Citations (Scopus)

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 languageEnglish (US)
Pages (from-to)745-750
Number of pages6
JournalMolecular Cell
Volume17
Issue number5
DOIs
StatePublished - Mar 4 2005

Fingerprint

Exodeoxyribonuclease V
Holoenzymes
Nanoparticles
Genetic Recombination
Escherichia coli
DNA
Enzymes

ASJC Scopus subject areas

  • 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 journalArticle

Handa, Naofumi ; Bianco, Piero R. ; Baskin, Ronald J. ; Kowalczykowski, Stephen C. / Direct visualization of RecBCD movement reveals cotranslocation of the RecD motor after χ recognition. In: Molecular Cell. 2005 ; Vol. 17, No. 5. pp. 745-750.
@article{64c9376e015a4936acbf181d49a7c15b,
title = "Direct visualization of RecBCD movement reveals cotranslocation of the RecD motor after χ recognition",
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.",
author = "Naofumi Handa and Bianco, {Piero R.} and Baskin, {Ronald J.} and Kowalczykowski, {Stephen C.}",
year = "2005",
month = "3",
day = "4",
doi = "10.1016/j.molcel.2005.02.011",
language = "English (US)",
volume = "17",
pages = "745--750",
journal = "Molecular Cell",
issn = "1097-2765",
publisher = "Cell Press",
number = "5",

}

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 -