Letter | Published:

Processive translocation and DNA unwinding by individual RecBCD enzyme molecules

Nature volume 409, pages 374378 (18 January 2001) | Download Citation

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Abstract

RecBCD enzyme is a processive DNA helicase1 and nuclease2 that participates in the repair of chromosomal DNA through homologous recombination3,4. We have visualized directly the movement of individual RecBCD enzymes on single molecules of double-stranded DNA (dsDNA). Detection involves the optical trapping of solitary, fluorescently tagged dsDNA molecules that are attached to polystyrene beads, and their visualization by fluorescence microscopy5,6. Both helicase translocation and DNA unwinding are monitored by the displacement of fluorescent dye from the DNA by the enzyme7. Here we show that unwinding is both continuous and processive, occurring at a maximum rate of 972 ± 172 base pairs per second (0.30 µm s-1), with as many as 42,300 base pairs of dsDNA unwound by a single RecBCD enzyme molecule. The mean behaviour of the individual RecBCD enzyme molecules corresponds to that observed in bulk solution.

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Acknowledgements

We would like to thank S. Chan and J. Lengyel for assistance with measurements, and the following people for their comments on the manuscript: N. Handa, J. Kleiman, A. Mazin, J. New, E. Seitz, M. Spies, T. Sugiyama and Y. Wu. This work was supported by an NIH Grant to S.C.K. and a DOE Center of Excellence for Laser Applications in Medicine Grant to Y.Y. and R.J.B.

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  1. Sections of

  2. *Microbiology and of

    • Piero R. Bianco
    •  & Stephen C. Kowalczykowski
  3. †Molecular and Cellular Biology and

    • Piero R. Bianco
    • , Stephen C. Kowalczykowski
    •  & Ronald J. Baskin
  4. Department of Applied Science, University of California at Davis, Davis, California 95616, USA

    • Yin Yeh
  5. ‡Electronics Engineering Technologies Division, and

    • Laurence R. Brewer
  6. §Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, California 94550, USA

    • Michele Corzett
    •  & Rod Balhorn

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Correspondence to Stephen C. Kowalczykowski.

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https://doi.org/10.1038/35053131

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