Forward and Reverse Motion of Single RecBCD Molecules on DNA

RecBCD is a processive, DNA-based motor enzyme with both helicase and nuclease activities. We used high-resolution optical trapping to study individual RecBCD molecules moving against applied forces up to 8 pN. Fine-scale motion was smooth down to a detection limit of 2 nm, implying a unitary step s...

Full description

Saved in:
Bibliographic Details
Published in:Biophysical journal 2004-03, Vol.86 (3), p.1640-1648
Main Authors: Perkins, Thomas T., Li, Hung-Wen, Dalal, Ravindra V., Gelles, Jeff, Block, Steven M.
Format: Article
Language:English
Subjects:
Binding Sites
Deoxyribonucleic acid
DNA
DNA - chemistry
Enzyme Activation
Enzyme Stability
Enzymes
Exodeoxyribonuclease V - chemistry
Kinetics
Lasers
Macromolecular Substances
Micromanipulation - methods
Molecular Motor Proteins
Molecules
Motion
Nucleic Acid Conformation
Nucleic Acid Denaturation
Nucleic Acids
Protein Binding
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:RecBCD is a processive, DNA-based motor enzyme with both helicase and nuclease activities. We used high-resolution optical trapping to study individual RecBCD molecules moving against applied forces up to 8 pN. Fine-scale motion was smooth down to a detection limit of 2 nm, implying a unitary step size below six basepairs (bp). Episodes of constant-velocity motion over hundreds to thousands of basepairs were punctuated by abrupt switches to a different speed or by spontaneous pauses of mean length 3 s. RecBCD occasionally reversed direction, sliding backward along DNA. Backsliding could be halted by reducing the force, after which forward motion sometimes resumed, often after a delay. Elasticity measurements showed that the DNA substrate was partially denatured during backsliding events, but reannealed concomitant with the resumption of forward movement. Our observations show that RecBCD-DNA complexes can exist in multiple, functionally distinct states that persist for many catalytic turnovers: such states may help tune enzyme activity for various biological functions.
ISSN:0006-3495
1542-0086
DOI:10.1016/S0006-3495(04)74232-0