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RecBCD enzyme is a DNA helicase with fast and slow motors of opposite polarity

Abstract

Helicases are molecular motors that move along and unwind double-stranded nucleic acids1. RecBCD enzyme is a complex helicase and nuclease, essential for the major pathway of homologous recombination and DNA repair in Escherichia coli2. It has sets of helicase motifs1 in both RecB and RecD, two of its three subunits. This rapid, highly processive enzyme unwinds DNA in an unusual manner: the 5′-ended strand forms a long single-stranded tail, whereas the 3′-ended strand forms an ever-growing single-stranded loop and short single-stranded tail. Here we show by electron microscopy of individual molecules that RecD is a fast helicase acting on the 5′-ended strand and RecB is a slow helicase acting on the 3′-ended strand on which the single-stranded loop accumulates. Mutational inactivation of the helicase domain in RecB or in RecD, or removal of the RecD subunit, altered the rates of unwinding or the types of structure produced, or both. This dual-helicase mechanism explains how the looped recombination intermediates are generated and may serve as a general model for highly processive travelling machines with two active motors, such as other helicases and kinesins.

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Figure 1: RecBK29QCD enzyme, with an inactive RecB helicase, unwinds DNA by means of a loop and a 5′-terminated tail.
Figure 2: Enzymes without RecD or with mutant RecD (RecBCDK177Q) unwind DNA slowly and with different topologies.
Figure 3: Analysis of DNA unwinding by RecBCD and RecBCDK177Q.
Figure 4: Model for unwinding by RecBCD enzyme.

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Acknowledgements

We thank D. Julin for advice on protein purification; M. Dillingham, M. Spies and S. Kowalczykowski for sharing their unpublished information7; S. Amundsen for permission to cite unpublished results; J. Cooper, M. Gellert, N. Maizels, R. Strong and our colleagues for comments on the manuscript; and L. Caldwell and staff for help with the electron microscopy. This research was supported by grants from the NIH.

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Correspondence to Gerald R. Smith.

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Taylor, A., Smith, G. RecBCD enzyme is a DNA helicase with fast and slow motors of opposite polarity. Nature 423, 889–893 (2003). https://doi.org/10.1038/nature01674

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