Processive translocation and DNA unwinding by individual RecBCD enzyme molecules

RecBCD enzyme is a processive DNA helicase and nuclease that participates in the repair of chromosomal DNA through homologous recombination. We have visualized directly the movement of individual RecBCD enzymes on single molecules of double-stranded DNA (dsDNA). Detection involves the optical trappi...

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Bibliographic Details
Published in:Nature (London) 2001-01, Vol.409 (6818), p.374-378
Main Authors: Kowalczykowski, Stephen C, Bianco, Piero R, Brewer, Laurence R, Corzett, Michele, Balhorn, Rod, Yeh, Yin, Baskin, Ronald J
Format: Article
Language:English
Subjects:
Adenosine Triphosphate - metabolism
Bacteriology
Biological and medical sciences
Biological Transport
Deoxyribonucleic acid
DNA
DNA - metabolism
DNA Helicases - metabolism
DNA, Viral
Enzymes
Exodeoxyribonuclease V
Exodeoxyribonucleases - metabolism
Fundamental and applied biological sciences. Psychology
Genetics
Image Processing, Computer-Assisted
Lasers
Microbiology
Microscopy, Fluorescence
Microscopy, Video
Molecular biology
Optics and Photonics
polystyrene
RecBCD enzyme
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Summary:RecBCD enzyme is a processive DNA helicase and nuclease that participates in the repair of chromosomal DNA through homologous recombination. 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 microscopy. Both helicase translocation and DNA unwinding are monitored by the displacement of fluorescent dye from the DNA by the enzyme. 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.
ISSN:0028-0836
1476-4687
DOI:10.1038/35053131