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 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.
Department of Biological Sciences, Stanford University, Stanford, California 94305-5020, USA. tperkins@jila.colorado.edu
Thomas T
TT
Perkins
Hung-Wen
HW
Li
Ravindra V
RV
Dalal
Jeff
J
Gelles
Steven M
SM
Block
D001665:Binding Sites; D004247:DNA / Q000737:chemistry*; D004789:Enzyme Activation; D004795:Enzyme Stability; D043211:Exodeoxyribonuclease V / Q000737:chemistry*; D007700:Kinetics; D007834:Lasers; D046911:Macromolecular Substances; D008846:Micromanipulation / Q000379:methods*; D020409:Molecular Motor Proteins; D009038:Motion; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D011485:Protein Binding
D046911:Macromolecular Substances; D020409:Molecular Motor Proteins; D004247:DNA; D043211:Exodeoxyribonuclease V