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Processive translocation and DNA unwinding by individual RecBCD enzyme molecules

Author

Listed:
  • Piero R. Bianco

    (Sections of Microbiology
    Sections of Molecular and Cellular Biology)

  • Laurence R. Brewer

    (Electronics Engineering Technologies Division)

  • Michele Corzett

    (Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory)

  • Rod Balhorn

    (Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory)

  • Yin Yeh

    (University of California at Davis)

  • Stephen C. Kowalczykowski

    (Sections of Microbiology
    Sections of Molecular and Cellular Biology)

  • Ronald J. Baskin

    (Sections of Molecular and Cellular Biology)

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.

Suggested Citation

  • Piero R. Bianco & Laurence R. Brewer & Michele Corzett & Rod Balhorn & Yin Yeh & Stephen C. Kowalczykowski & Ronald J. Baskin, 2001. "Processive translocation and DNA unwinding by individual RecBCD enzyme molecules," Nature, Nature, vol. 409(6818), pages 374-378, January.
    • Handle: RePEc:nat:nature:v:409:y:2001:i:6818:d:10.1038_35053131
    DOI: 10.1038/35053131
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