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Direct observation of DNA translocation and cleavage by the EcoKI endonuclease using atomic force microscopy

Nature Structural Biology volume 6, pages 15–17 (1999)Cite this article

Type I DNA restriction and modification enzymes such as EcoKI are multimeric enzymes that cleave DNA molecules lacking an appropriate pattern of methylation on a target sequence, thereby protecting the host bacterium from invasion by foreign DNA1,2,3,4,5. The reaction involves the initial recognition of an unmethylated DNA target sequence with the aid of the cofactor S-adenosyl methionine. Recognition is followed by extensive ATP-dependent translocation of the DNA in both directions toward the enzyme, which remains bound at the target sequence. Eventually, endonucleolytic cleavage of the DNA occurs6,7. Recently, protein complexes with nucleic acids have been imaged successfully using fluid tapping-mode atomic force microscopy (AFM)8,9,10,11. In this study, we have used AFM to explore the type I DNA restriction and modification pathway5, and specifically to follow the ATP-dependent translocation and cleavage of a single plasmid by the EcoKI enzyme complex under near-physiological conditions.

EcoKI free in solution is ∼437,000 Mr5 and has the subunit stoichiometry R2M2S1, where R, M and S are the restriction endonuclease/helicase, modification methyltransferase and sequence-specificity subunits, respectively. In isolation, EcoKI appeared as a homogeneous population of randomly arranged particles (data not shown). Analysis of cross sections of the particles yields a diameter of 21.0 ± 0.6 nm and a height of 3.1 ± 0.1 nm (n = 19, mean ± standard error of the mean) Using these dimensions, the molecular volume of the particle was estimated as described12. The value obtained, 556 nm3, is close to the volume derived from the expected molecular weight (747 nm3), indicating that the enzyme behaves as a monomeric complex in the absence of DNA.

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Figure 1: Images of pRH3 plasmids and complexes of EcoKI with pRH3 and pBRSK15.
Figure 2: Translocation and cleavage of pRH3 by EcoKI.

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Acknowledgements

We thank G. Davies and N. Murray (University of Edinburgh) for their kind gift of E. coli NM679 and plasmid pBRSK15, and D. Hornby (University of Sheffield) for fruitful discussions. This work was funded by the Biotechnology and Biological Sciences Research Council (D.J.E., J.M.E., R.M.H.), and the Royal Society (D.T.F.D.). T.B. holds a European Community TMR Marie Curie Award. David Dryden wishes to thank the Royal Society for a University Research Fellowship.

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  1. Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QJ, UK

    Darren J. Ellis, Torunn Berge, J. Michael Edwardson & Robert M. Henderson

  2. Institute of Cell and Molecular Biology, The University of Edinburgh, Darwin Building, The King's Buildings, Mayfield Road, Edinburgh, EH9 3JR, UK

    David T.F. Dryden

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Correspondence to Robert M. Henderson.

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Ellis, D., Dryden, D., Berge, T. et al. Direct observation of DNA translocation and cleavage by the EcoKI endonuclease using atomic force microscopy. Nat Struct Mol Biol 6, 15–17 (1999). https://doi.org/10.1038/4882

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