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Forward and Reverse Motion of Single RecBCD Molecules on DNA

Thomas T. Perkins ^*, Hung-Wen Li , Ravindra V. Dalal ^*, Jeff Gelles  and Steven M. Block ^*

^* Departments of Biological Sciences and Applied Physics, Stanford University, Stanford, California 94305-5020; and Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02454-9110

Correspondence: Address reprint requests to Thomas T. Perkins, E-mail: tperkins{at}jila.colorado.edu.

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.

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