Fig. 2.

DNA cleavage activity of motif II and III nuclease mutants of EcoR124I. (a) Comparison of cleavage end points of WT and mutant HsdR subunits. Agarose gel electrophoresis was used to analyse the single-site substrate pCFD30 that was incubated for 5 min at 37 °C with saturating amounts of MTase (20 nM) and HsdR (160 nM) in the presence of 4 mM ATP (Materials and Methods). CCC is covalently closed circular DNA (substrate), OC is open circle DNA (product cut in one strand) and FLL is full-length linear DNA (product cut in both strands). (b and c) Comparison of the cleavage time course for WT HsdR (b) and E165D HsdR (c) at 25 °C. pMDS27.3 and saturating MTase (40 nM) and HsdR (100 nM) were preincubated, and the reaction was initiated by the addition of ATP to 4 mM. Samples were removed at the time points indicated and immediately quenched. The DNA substrate and products were then separated by agarose gel electrophoresis and quantified by scintillation counting (Materials and Methods). The different relative mobilities of the FLL fragments reflect different running conditions for each gel (i.e., different values in V cm^− 1). DNA dimers are a minor contaminant in our plasmid preparations. (d) Fitting of the CCC data from WT (b), E165D (c) and E165A (data not shown) using Eq. (1) as described in Materials and Methods. The fitted parameters are given in Table 2. Under our reaction conditions, DNA cleavage by EcoR124I never goes to completion because of a background inhibition activity that competes with the translocation/cleavage process. Regardless of the observed cleavage rates, very little change in the relative levels of the species is observed beyond ∼ 5–10 min. This has been noted previously with EcoR124I using both WT enzyme and QxxxY motif mutants⁸ and has also been observed with EcoKI, but not with EcoAI (F. Peske, unpublished observations). The effect appears to be dependent on changes to the DNA that make it resistant and is not dependent on inhibition of the protein. However, the exact nature of the inhibition of DNA cleavage is unclear and is currently still under investigation (F. Peske, unpublished observations).