Nucleic Acids Research, 2010, Vol. 38, No. 19 6593 Figure 2. Strand cleavage and type I endonuclease activities of Cre(R173A) on an MeP-half-site substrate. In the schematic representation of the half-site substrate, the asterisk indicates 32P-label at the 50-end, and ‘mp’ the scissile methylphosphonate. The parallel arrows denote the Cre binding element. Short stretches of additional nucleotides extending from the binding element to its left on both strands are not shown. The type I endonuclease activity proceeds via formation of the MeP–tyrosyl intermediate, followed by its hydrolysis. Reactions were split into halves to analyze the covalent Cre–DNA adduct (indicated by the circular knob linked to the labeled DNA chain) by SDS–PAGE (A) and the potential hydrolysis product(s) by denaturing urea–PAGE (B). The end-labeled half-site (A) or its labeled strand (B) is denoted by ‘S’, and the hydrolysis product by ‘HP’. The labeled strand in the MeP-half-site is 27-nt long, and HP is a 24-mer. In the control reaction, the MeP-half-site was incubated in the recombination buﬀer for 48 h in the absence of Cre(R173A). The plots represent mean values from three separate experiments with Cre(R173A). The global ﬁt to the kinetic data was performed using KinTek Global Kinetic Explorer, as explained in ‘Materials and Methods’ section. For a given starting concentration of the MeP-half-site, each rate constant and the output scaling factor were adjusted to obtain the optimal global ﬁt. Reactions with Cre(R173A) revealed the early forma- tion of the MeP–tyrosyl intermediate (Figure 2A), followed by its subsequent decay. The 24-mer hydrolysis product was also formed in the reaction but with a time delay compared to the tyrosyl adduct (Figure 2B). The correspondence between the diminution of this adduct and the build-up of the 24-mer was consistent with a pre- cursor–product relationship between the two. Furthermore, in agreement with this notion, blocking strand cleavage by eliminating the Tyr-324 nucleophile also blocked 24-mer formation. Neither the covalent cleaved intermediate (Figure 3A) nor the 24-mer (Figure 3B) was detected in a reaction with Cre(R173A, Y324G). The mobility of the major 24-mer product cor- responded to the presence of MeP at its 30 end, while that of the faint band just above indicated a 30-hydroxyl end (7). Cre(R173A) was inactive on a P-half-site (Figure 4). Thus, the absence of Arg-173 or that of Arg-292 in Cre does not render the scissile MeP bond vulnerable to direct hydrolysis. However these individual mutations do not prevent MeP activation and formation of the MeP– tyrosyl intermediate. The strand cleavage step during Cre recombination is presumably protected by the mono- meric cis-organization, rather than the electrostatic properties, of its active site. The ﬁrst order rate constants for the strand cleavage and type I endonuclease activities of Cre(R173A) were estimated as kcl = 4.4 10 4 s 1 and khydrol = 2.7 10 4 s 1, respectively (Table 2). Unlike the situation with topoisomerase, hydrolysis of the MeP–tyrosyl inter- mediate is not dramatically accelerated by Cre(R173A) or Cre(R292A) relative to that of the P–tyrosyl intermediate by wild-type Cre (khydrol = 2.8 10 5 s 1) (7). The corres- ponding rate constants for topoisomerase diﬀer by a factor of >104 between the MeP- and P–tyrosyl intermedi- ates (9). Strand cleavage and type I endonuclease activities of Cre(R173A, R292A) In addition to the conserved twin arginines, the Cre cata- lytic pentad contains additional positive charges in the form of Lys-201 and His-289. Thus, there is a cradle of positive charge within the active site that presumably adds a signiﬁcant electrophilic character to the chemical steps of recombination (2,3). In the crystal structure, His-289 forms hydrogen bonding contact with the scissile phos- phate after it has been cleaved but not prior to cleavage (2) (Figure 1). Trp-315 of the pentad is within hydrogen bonding distance of the scissile phosphate in both its uncleaved and cleaved states (Figure 1). We wondered whether, even in the combined absence of Arg-173 and Arg-292, MeP activation might be feasible through the residual positive charge in the active site and/or the hydrogen bonding interactions through His-289 and Trp-315. A related question, assuming that Cre(R173A, R292A) promotes MeP activation, is whether the double mutation might cause susceptibility of MeP to direct hy- drolysis (type II endonuclease activity). Cre(R173A, R292A) not only produced the covalent intermediate in the MeP-half-site but, rather surprisingly, the initial rate of its formation was similar to that observed in the Cre(R173A) reaction (Figure 5A). The