Initiation of translocation by Type I restriction-modification enzymes is associated with a short DNA extrusion

Recognition of ‘foreign’ DNA by Type I restriction–modification (R-M) enzymes elicits an ATP-dependent switch from methylase to endonuclease activity, which involves DNA translocation by the restriction subunit HsdR. Type I R-M enzymes are composed of three (Hsd) subunits with a stoichiometry of HsdR₂:HsdM₂:HsdS₁ (R₂-complex). However, the EcoR124I R-M enzyme can also exist as a cleavage deficient, sub-assembly of HsdR₁:HsdM₂:HsdS₁ (R₁-complex). ATPγS was used to trap initial translocation complexes, which were visualized by Atomic Force Microscopy (AFM). In the R₁-complex, a small bulge, associated with a shortening in the contour-length of the DNA of 8 nm, was observed. This bulge was found to be sensitive to single-strand DNA nucleases, indicative of non-duplexed DNA. R₂-complexes appeared larger in the AFM images and the DNA contour length showed a shortening of ~11 nm, suggesting that two bulges were formed. Disclosure of the structure of the first stage after the recognition-translocation switch of Type I restriction enzymes forms an important first step in resolving a detailed mechanistic picture of DNA translocation by SF-II DNA translocation motors.