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Forward and reverse motion of single RecBCD molecules on DNA
Forward and reverse motion of single RecBCD molecules on DNA.
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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.
Perkins TT, Li HW, Dalal RV, Gelles J, Block SM
Biophysical journal
2004-03-01 00:00
86
3
1640-8
Binding Sites,DNA,Enzyme Activation,Enzyme Stability,Exodeoxyribonuclease V,Kinetics,Lasers,Macromolecular Substances,Micromanipulation,Molecular Motor Proteins,Motion,Nucleic Acid Conformation,Nucleic Acid Denaturation,Protein Binding,Macromolecular Substances,Molecular Motor Proteins,DNA,Exodeoxyribonuclease V
Department of Biological Sciences, Stanford University, Stanford, California 94305-5020, USA tperkinsjilacoloradoedu
Biophys. J.
NIGMS R01 GM057035-09
0006-3495
1000
True
14990491
