Nucleic Acids Research, 2010, Vol. 38, No. 19 6595 Table 2. Activities of Cre mutants containing alanine replacements at Arg-173 or Arg-292 or both positions deduced from this study are summarized Cre mutant Cre(R173A) Cre(R292A) Activities on MeP DNA substrates Strand cleavage Type I endonuclease (hydrolysis of MeP–tyrosyl intermediate) Type II endonuclease Strand joining (direct hydrolysis of MeP bond in DNA) Recombination Active (kcl = 4.4 10 4s 1) Active (khydrol = 2.7 10 4s 1) Inactive Active (kcl = 3.2 10 4s 1) Active (kcl = 8.8 10 4s 1) Inactive Inactive Active (kjoin = 8.6 10 6s 1) Active (kjoin = 2.8 10 5s 1) Inactive Active Active Inactive Cre(R173A, R292A) Active ðk1Cl¼ 3:2 10 4s 1 k2Cl¼ 1:7 10 4s 1Þ Active ðk1hydrol¼ 7:8 10 4s 1 k2hydrol¼ 5:1 10 5s 1Þ The kinetic data for the strand cleavage and endonuclease activities of Cre(R173A, R292A) yielded better global ﬁt by assuming two contributing ﬁrst-order rate constants for each activity. The distinct reactivities of the RP and SP forms of the MeP–substrate (provided as an achiral mixture of the two forms) could potentially account for the binary rate constants. Figure 5. Strand cleavage and endonuclease activities of Cre(R173A, R292A) on an MeP-half-site. Reactions and analyses were carried out as described under Figure 2. In the control reaction, the MeP-half-site was incubated without Cre(R173A, R292A) in the reaction buﬀer for 24 h. The values from three separate experiments are plotted in the graph. Assumption of two contributing ﬁrst order rate constants, rather than a single rate constant, for the strand cleavage and type I endonuclease reactions gave better global ﬁt to the experimental data. (A) SDS-PAGE. (B) Urea-PAGE. MeP–tyrosyl intermediate was longer lived in the double mutant reaction, peaking at 4 h. Its disappearance with concomitant formation of the 24-mer hydrolysis product (via the type I endonuclease mechanism) did not become signiﬁcant until 6–8 h from start (Figure 5B). A potential type II endonuclease activity of Cre(R173A, R292A) was ruled out by incorporating into it the Y324G mutation as well. The triple mutant was unable to generate the covalent intermediate as expected, or to promote direct MeP hydrolysis (Figure 6A and B). The kinetic data in Figure 5 could not be easily accommodated by an irreversible two-step reaction model, with a single rate constant for each step. A better ﬁt was obtained by introducing two contributing rate constants for strand cleavage ðk1Cl¼ 3:2 10 4s 1; k2Cl ¼k2hy1d:r7ol¼ 150:1 4s 110Þ 5asnd1Þ.hyOdnroelypsoisssðibk1hilyidtryol¼is 7:t8hat 1t0he4sR1P; and SP forms present in the achiral mixture of the MeP-half-site are kinetically distinct in their reactions with Cre(R173A, R292A). A previous study (7) showed that Cre(R292A) has a strong bias favoring SP over RP. The stereochemical preference of Cre(R173A) or Cre(R173A, R292A) has not yet been determined. Strand joining by Cre(R173A), Cre(R292A) and Cre(R173A, R292A) The strand cleavage potential of the single and double alanine substitution mutants of Arg-173 and Arg-292