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  • The EMBO Journal (2005) 24, 4188 - 4197
  • doi:10.1038/sj.emboj.7600881

Published online: 17 November 2005

Dynamics of initiation, termination and reinitiation of DNA translocation by the motor protein EcoR124I

Ralf Seidel1, Joost GP Bloom1, John van Noort1,a, Christina F Dutta2, Nynke H Dekker1, Keith Firman2, Mark D Szczelkun3 and Cees Dekker1

  1. Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands
  2. School of Biological Sciences, University of Portsmouth, Portsmouth, UK
  3. Department of Biochemistry, School of Medical Sciences, Bristol, UK

Correspondence to:

Mark D Szczelkun, Department of Biochemistry, School of Medical Sciences, University Walk, Bristol BS8 1TD, UK. Tel.: +44 117 928 7439; Fax: +44 117 928 8274; E-mail: mark.szczelkun@bristol.ac.uk

Cees Dekker, Kavli Institute of Nanoscience, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands. Tel.: +31 15 278 6094; Fax: +31 15 278 1202; E-mail: dekker@mb.tn.tudelft.nl

aPresent address: Biophysics, LION, Leiden University, Niels Bohrweg 2, 2333 RA Leiden, The Netherlands

Received 1 July 2005; Accepted 28 October 2005

Type I restriction enzymes use two motors to translocate DNA before carrying out DNA cleavage. The motor function is accomplished by amino-acid motifs typical for superfamily 2 helicases, although DNA unwinding is not observed. Using a combination of extensive single-molecule magnetic tweezers and stopped-flow bulk measurements, we fully characterized the (re)initiation of DNA translocation by EcoR124I. We found that the methyltransferase core unit of the enzyme loads the motor subunits onto adjacent DNA by allowing them to bind and initiate translocation. Termination of translocation occurs owing to dissociation of the motors from the core unit. Reinitiation of translocation requires binding of new motors from solution. The identification and quantification of further initiation steps—ATP binding and extrusion of an initial DNA loop—allowed us to deduce a complete kinetic reinitiation scheme. The dissociation/reassociation of motors during translocation allows dynamic control of the restriction process by the availability of motors. Direct evidence that this control mechanism is relevant in vivo is provided.

  • Keywords:

    • DNA translocation,
    • molecular motor,
    • restriction enzymes,
    • SF2 helicase,
    • single molecules