Initiation of Translocation

Type I R‑M enzymes are composed of three (Hsd) subunits with a Stoichiometry of HsdR₂:HsdM₂:HsdS₁ (R₂‑complex), a core DNA-binding MTase (M₂S₁) binds to the recognition sequence and the two HsdR subunits bind to the MTase (Figure 2). However, the EcoR124I R-M enzyme can also exist as a cleavage deficient, sub-assembly of HsdR₁:HsdM₂:HsdS₁ (R₁‑complex).  ATPgS 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 (van Noort et al. 2004)(Figure 3). 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 around 11 nm, suggesting that two bulges were formed.