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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41586_2001_BF35053131_MOESM1_ESM.mov
This movie shows unwinding of a single molecule of lambda DNA by a simgle RecBCD enzyme at 37°C in the presence of 1mM ATP. (MOV 3468 kb)
41586_2001_BF35053131_MOESM2_ESM.mov
This movie shows unwinding of a single molecule of lambda DNA by a simgle RecBCD enzyme at 23°C in the presence of 1mM ATP. (MOV 3622 kb)
41586_2001_BF35053131_MOESM3_ESM.mov
This movie shows unwinding of a single molecule of lambda DNA by a simgle RecBCD enzyme at 23°C in the presence of 250μM ATP. (MOV 3588 kb)
41586_2001_BF35053131_MOESM5_ESM.jpg
A graph showing the amount of active protein in the RecBCD enzyme prepatation used in the experiments described. (JPG 99 kb)
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Bianco, P., Brewer, L., Corzett, M. et al. Processive translocation and DNA unwinding by individual RecBCD enzyme molecules. Nature 409, 374–378 (2001). https://doi.org/10.1038/35053131
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DOI: https://doi.org/10.1038/35053131
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