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Using a combination of single molecule and bulk solution measurements, we have examined the DNA translocation activity of a helicase, the Type I restriction modification enzyme EcoR124I. We find that EcoR124I can translocate past covalent interstrand crosslinks, inconsistent with an obligatory unwinding mechanism. Instead, translocation of the intact dsDNA occurs principally via contacts to the sugar-phosphate backbone and bases of the 3'-5' strand; contacts to the 5'-3' strand are not essential for motion but do play a key role in stabilising the motor on the DNA. A model for dsDNA translocation is presented that could be applicable to a wide range of other enzyme complexes that are also labelled as helicases but which do not have actual unwinding activity.

Type I restriction enzymes bind sequence-specifically to unmodified DNA and subsequently pull the adjacent DNA toward themselves. Cleavage then occurs remotely from the recognition site. The mechanism by which these members of the superfamily 2 (SF2) of helicases translocate DNA is largely unknown. We report the first single-molecule study of DNA translocation by the type I restriction enzyme EcoR124I. Mechanochemical parameters such as the translocation rate and processivity, and their dependence on force and ATP concentration, are presented. We show that the two motor subunits of EcoR124I work independently. By using torsionally constrained DNA molecules, we found that the enzyme tracks along the helical pitch of the DNA molecule. This assay may be directly applicable to investigating the tracking of other DNA-translocating motors along their DNA templates.