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χ-Sequence recognition and DNA translocation by single RecBCD helicase/nuclease molecules

Nature volume 409, pages 370–374 (2001)Cite this article

Abstract

Major pathways of recombinational DNA repair in Escherichia coli require the RecBCD protein—a heterotrimeric, ATP-driven, DNA translocating motor enzyme. RecBCD combines a highly processive and exceptionally fast helicase (DNA-unwinding) activity with a strand-specific nuclease (DNA-cleaving) activity (refs 1, 2 and references therein). Recognition of the DNA sequence ‘χ’ (5′-GCTGGTGG-3′) switches the polarity of DNA cleavage and stimulates recombination at nearby sequences in vivo. Here we attach microscopic polystyrene beads to biotin-tagged RecD protein subunits and use tethered-particle light microscopy to observe translocation of single RecBCD molecules (with a precision of up to ∼30 nm at 2 Hz) and to examine the mechanism by which χ modifies enzyme activity. Observed translocation is unidirectional, with each molecule moving at a constant velocity corresponding to the population-average DNA unwinding rate. These observations place strong constraints on possible movement mechanisms. Bead release at χ is negligible, showing that the activity modification at χ does not require ejection of the RecD subunit from the enzyme as previously proposed; modification may occur through an unusual, pure conformational switch mechanism.

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Figure 1: Observing translocation along DNA by single RecBCD-bio molecules.
Figure 2: Time courses and rates of translocation.
Figure 3: Non-denaturing electrophoresis gel autoradiogram showing production of χ-specific DNA fragments by RecBCD-bio.
Figure 4: RecD release at χ.

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Acknowledgements

We thank P. Bianco, P. Boehmer, S. Kowalczykowski, S. Lovett and A. Taylor for materials and helpful advice. This work was supported by the NIGMS.

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Authors and Affiliations

  1. Department of Biochemistry, Brandeis University, Waltham , 02454-9110, Massachusetts, USA

    Kathleen M. Dohoney & Jeff Gelles

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  1. Kathleen M. Dohoney
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Correspondence to Jeff Gelles.

Supplementary information

41586_2001_BF35053124_MOESM1_ESM.mov

Quicktime video of a bead-labeled RecBCD-bio molecule translocating along a single DNA molecule. This experiment was performed as shown in Fig. 1 of the paper, except that the ATP concentration was reduced to 10 µM to slow translocation. The video is real time; the frame size is 6.5 µm wide by 6.6 µm tall. (MOV 1293 kb)

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Dohoney, K., Gelles, J. χ-Sequence recognition and DNA translocation by single RecBCD helicase/nuclease molecules. Nature 409, 370–374 (2001). https://doi.org/10.1038/35053124

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