6592 Nucleic Acids Research, 2010, Vol. 38, No. 19 gel electrophoresis (SDS–PAGE). For assaying hydroly- sis, DNA was isolated by phenol–chloroform extraction followed by ethanol precipitation at 20 C. The DNA samples were washed twice with 80% ethanol, and analyzed by 12% urea–PAGE. In this assay, the protein- ase K treatment that normally precedes DNA extraction was omitted. By doing so, following electrophoresis, the hydrolysis product could be visualized without interfer- ence from the strand cleavage product carrying short Cre peptides at the 30 end. DNA samples isolated from recombination reactions between two full-LoxP-sites were also analyzed by 12% urea–PAGE. Kinetic analysis of the type I hydrolysis reaction by Cre(R173A) and Cre(R173A, R292A) Kinetic parameters were derived according to a two-step reaction scheme, strand cleavage by the tyrosine nucleo- phile of Cre followed by hydrolysis of the DNA–tyrosyl intermediate. Cre+½S ½Cre kcl ½Cre S1 +S2 ! S1 khydrol Cre+S1 ! S is the substrate, and kcl and khydrol are the ﬁrst order rate constants for cleavage and hydrolysis, respectively. Cre-S1 refers to the covalent adduct, S2 to the trinucleotide released during strand cleavage and S1 to the hydrolysis product. Each of the two reaction steps is assumed to be irreversible. Curve ﬁtting by non-linear regression was performed by using KinTek Global Kinetic Explorer simulation/dynamic visualization software (18,19). For a given starting concentration of S, each rate constant and the output scaling factor were adjusted to obtain the optimal global ﬁt to the experimental data. Kinetic analysis of strand joining by Cre(R173A) and Cre(292A) The ﬁrst order rate constants for the strand joining reaction were estimated according to the scheme: Cre+½S ½Cre kcl ½Cre S1 +S2 ! S1 kjoin Cre+SJP ! SJP denotes the product of strand joining. Curve ﬁtting was performed as described above for the strand cleavage and hydrolysis reactions. Quantiﬁcation of reaction products Gels in which reactions were analyzed were dried and exposed to a storage phosphor screen (Bio-Rad) or X-ray ﬁlm to detect radioactive bands. Exposure times were optimized to obtain reliable signals while avoiding saturation of the screen. Phosphor screens were scanned by a phosphor-imager (GE-Healthcare). Image analysis was performed by using the software Quantity One (version 4.5.1; Bio-Rad). RESULTS Experimental layout Assays were carried out primarily using MeP substrates (7), in the form of half-LoxP or full-LoxP sites, supplied as a racemic mixture of the RP and SP stereoisomers. A subset of reactions were performed on P substrates as controls. A half-site contains only one Cre-binding element and one scissile strand. When the Tyr-324 nucleo- phile performs strand cleavage in a half-site, the short trinucleotide product of the reaction (with a 50-hydroxyl group) will not remain stably hydrogen bonded to the opposite strand. The diﬀusion of the trinucleotide away from the reaction center makes the cleavage reaction ef- fectively irreversible. If the non-scissile strand of the half-site harbors a 50-hydroxyl end, a pseudo-joining reaction may occur by the attack of the hydroxyl on the cleaved tyrosyl intermediate. In principle, a scissile P- or MeP bond in the DNA substrate activated by a bound Cre or the corresponding tyrosyl intermediate formed by strand cleavage could be hydrolyzed as a result of nucleo- philic attack by water. In the experiments described below, strand cleavage, strand joining and potential hydrolytic reactions were assayed. Even though the Cre active site is contained within a monomer, its full chemical compe- tence may depend on allosteric activation by a second Cre monomer. Thus, the half-site reactions may occur in the context of dimeric, trimeric or tetrameric [Cre-half-site] complexes (20,21). In reactions that monitored hydrolysis promoted by a Cre mutant, the 50-hydroxyl of the strand complementary to the scissile one was blocked by phosphorylation. This hydroxyl was left unmodiﬁed when assaying strand joining. The covalent tyrosyl adduct formed by strand cleavage was revealed by SDS–PAGE. The products of hydrolysis, strand joining and recombination were visualized by electrophoresis under strand denaturing con- ditions (urea–PAGE). The substrates used in the MeP-full-site reaction contained a symmetrized 6-bp strand exchange region. Schematic representations of speciﬁc reactions are included in ﬁgures for clarity. Strand cleavage in the MeP-half-site by Cre(R173A): hydrolysis of the MeP–tyrosyl intermediate In previous work, we found that unlike Flp(R308A), the equivalent Cre(R292A) does not support direct hydrolysis of the MeP bond (type II endonuclease activity) (7). Instead, it promotes formation of the MeP–tyrosyl inter- mediate, which may then be targeted for hydrolysis by the type I endonuclease activity. The suggested explanation is that the cis-acting active site of a Cre monomer fully engages the scissile P or MeP upon DNA binding, thus excluding water from the reaction center. An alternative possibility is that it is Arg-173 of Cre (corresponding to Arg-191 of Flp), which also contacts the scissile phosphate (2) (Figure 1), that is responsible for repelling water during strand cleavage by Cre. We wished to know whether the R173A mutation in Cre would induce it to exhibit the type II endonuclease activity.