AFM Analysis of Translocation by EcoR124I

Peter Coxhead1, Christina Dutta1, James Smith1, Olga Kazakova2, John Van Noort3, Thijn Van Der Heijden4, Cees Dekker4 and Keith Firman1
1    University of Portsmouth, King Henry Building, King Henry I Street, Portsmouth Hants, PO1 2DY
2    National Physical Laboratory, Teddington, UK.
3    Leiden University, The Netherlands.
4    TUDelft, The Netherlands.

Abstract

Studies with EcoKI, the archetypical Type IA Restriction-Modification system, have suggested dimerisation is an integral part of the DNA translocation/cleavage mechanism.  However single molecule analysis of translocation by the Type IC R-M system EcoR124I shows no evidence for this effect.  We have also used the AFM to analyse the initiation of translocation.  ATPgS allows us to ‘trap’ the initial complex and close analysis of images obtained with the R1-complex of EcoR124I revealed a small bulge.  We will describe the possible nature of this bulge and its role in the translocation mechanism.

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