Figure 3. Strand cleavage and endonuclease activities of Cre in P- and MeP-half-site substrates.

In the schematic representations of half-site substrates, the asterisk indicates ³²P-label at the 5′-end, ‘p’ the scissile phosphate and ‘mp’ the scissile methylphosphonate. Reactions were split into halves to analyze the covalent Cre-DNA adduct (line ending in a circular knob) by SDS-PAGE (A) and the potential hydrolysis product(s) by denaturing PAGE (B). Reactions of Flp with its cognate half-sites in similar assays [19] are provided for comparison. The end-labeled half-site (A) or its labeled strand (B) is denoted by ‘S’ and the hydrolysis product by ‘HP’. The labeled strands in the Cre and Flp half-sites were 27 and 26 nucleotides long, respectively. HP in the Cre reaction was a 24-mer and that in the Flp reaction a 23-mer. The plots below represent mean values from three separate experiments with Cre.