Loading Dynamics of a Sliding DNA Clamp

Sliding DNA clamps are loaded at a ss/dsDNA junction by a clamp loader that depends on ATP binding for clamp opening. Sequential ATP hydrolysis results in closure of the clamp so that it completely encircles and diffuses on dsDNA. We followed events during loading of an E. coli β clamp in real time...

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Bibliographic Details
Published in:Angewandte Chemie International Edition 2014-06, Vol.53 (26), p.6768-6771
Main Authors: Cho, Won-Ki, Jergic, Slobodan, Kim, Daehyung, Dixon, Nicholas E., Lee, Jong-Bong
Format: Article
Language:English
Subjects:
Adenosine Triphosphate - chemistry
Adenosine Triphosphate - metabolism
Carbocyanines - chemistry
Clamps
Closures
Communications
Deoxyribonucleic acid
DNA - chemistry
DNA - metabolism
DNA clamps
DNA replication
Dynamics
Escherichia coli - metabolism
Escherichia coli Proteins - metabolism
Fluorescence Resonance Energy Transfer
Fretting
Hydrolysis
Loaders
Loads (forces)
Polarization
single-molecule FRET
single-molecule polarization
Sliding
ternary complexes
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Summary:Sliding DNA clamps are loaded at a ss/dsDNA junction by a clamp loader that depends on ATP binding for clamp opening. Sequential ATP hydrolysis results in closure of the clamp so that it completely encircles and diffuses on dsDNA. We followed events during loading of an E. coli β clamp in real time by using single‐molecule FRET (smFRET). Three successive FRET states were retained for 0.3 s, 0.7 s, and 9 min: Hydrolysis of the first ATP molecule by the γ clamp loader resulted in closure of the clamp in 0.3 s, and after 0.7 s in the closed conformation, the clamp was released to diffuse on the dsDNA for at least 9 min. An additional single‐molecule polarization study revealed that the interfacial domain of the clamp rotated in plane by approximately 8° during clamp closure. The single‐molecule polarization and FRET studies thus revealed the real‐time dynamics of the ATP‐hydrolysis‐dependent 3D conformational change of the β clamp during loading at a ss/dsDNA junction. When one clamp closes…︁ Single‐molecule Förster resonance energy transfer and single‐molecule fluorescence polarization were used to monitor the loading of a sliding DNA clamp onto double‐stranded DNA by the clamp loader (see picture). Thus, the dynamic features of a DNA clamp in the DNA/DNA‐clamp/clamp‐loader ternary complex were revealed.
ISSN:1433-7851
1521-3773
DOI:10.1002/anie.201403063