First-order rate constants for strand cleavage in P- and MeP-half-sites and hydrolysis of the cleaved intermediate by Cre and Cre(R292A).

The rate constants for strand cleavage (kcl) and hydrolysis of the tyrosyl intermediate (khydrol) were estimated from data shown in Figures 3 and 4 and additional data from similar assays. The software package Prism (version 5.02) for Windows (GraphPad Software, Inc.) was used for obtaining the kinetic parameters. In the reaction scheme

the first step of strand cleavage and formation of the Cre-DNA adduct is assumed to be irreversible. The trinucleotide product resulting from cleavage would diffuse away, and be unavailable for the back reaction. The values for the rate constants for Flp and vaccinia topoisomerase are taken from published work [19], [20].