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Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks

Abstract

RecBCD is a multi-functional enzyme complex that processes DNA ends resulting from a double-strand break. RecBCD is a bipolar helicase that splits the duplex into its component strands and digests them until encountering a recombinational hotspot (Chi site). The nuclease activity is then attenuated and RecBCD loads RecA onto the 3′ tail of the DNA. Here we present the crystal structure of RecBCD bound to a DNA substrate. In this initiation complex, the DNA duplex has been split across the RecC subunit to create a fork with the separated strands each heading towards different helicase motor subunits. The strands pass along tunnels within the complex, both emerging adjacent to the nuclease domain of RecB. Passage of the 3′ tail through one of these tunnels provides a mechanism for the recognition of a Chi sequence by RecC within the context of double-stranded DNA. Gating of this tunnel suggests how nuclease activity might be regulated.

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Figure 1: The processing of double-strand breaks by RecBCD enzyme.
Figure 2: Structures of the individual RecBCD subunits.
Figure 3: Structure of the RecBCD–DNA complex.
Figure 4: Alternative exits from the 3′ tunnel.
Figure 5: Diagram outlining the changes in RecBCD that occur after encountering a Chi site.

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Acknowledgements

We thank V. Ramakrishnan and J. Lowe for advice on the use and supply of tantalum bromide clusters, S. Halford for discussions about nucleases, C. Flensburg for advice on using SHARP and a pre-release version of the program, V. Dearing for oligonucleotide synthesis and purification, and D. Hall and E. Gordon for time and assistance on ESRF beamlines. This work was supported by Cancer Research UK (D.B.W.), NIH (S.C.K.) and a Wellcome Trust Travelling Research Fellowship (M.S.D.).

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Correspondence to Dale B. Wigley.

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Singleton, M., Dillingham, M., Gaudier, M. et al. Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks. Nature 432, 187–193 (2004). https://doi.org/10.1038/nature02988

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