R292A) did not give detectable joining reaction (Figure 7C). For comparison, in a P-half-site reaction with Cre, 50% of the input substrate was converted to the joined product in about 6 h (Figure 7D). For Cre(R173A) and Cre(R292A) reactions, the joined product was formed in signiﬁcantly higher yields than the 24-mer hydrolysis product, which was reduced to near background levels (Figure 7A and B). This prominence of strand joining relative to hydrolysis was also true of the wild-type Cre reaction with the P-half-site (Figure 7D). Thus, the 50-hydroxyl group eﬀectively out-competes water in nucleophilic attack on the MeP– tyrosyl or P–tyrosyl intermediate. The yields of strand joining by Cre(R173A) (Figure 7A) and Cre(R292A) (Figure 7B) were considerably dimin- ished in comparison to those of hydrolysis by these mutants from the MeP-half-site with the shorter bottom strand lacking the 50-hydroxyl nucleophile (7) (Figure 2A). The reduction was from 30–40% hydrolysis to 6% or less joining (and practically no hydrolysis) in the relevant substrates. It is possible that the extended bottom strand tail of the half-site, with its ability to fold back, and/or the 50-hydroxyl group could interfere with Tyr-324 mediated cleavage, lowering the levels of the tyroysl intermediate available for either hydrolysis or joining. Alternatively, Nucleic Acids Research, 2010, Vol. 38, No. 19 6597 lack of Arg-173 or Arg-292 might reduce the eﬃciency of strand joining, with the 50-hydroxyl group positioned within the active site additionally blocking nucleophilic attack by water due to steric interference. To test these possibilities, we assayed the extent of covalent adduct for- mation by each of the Cre mutants in the joining substrate. In Cre(R173A), Cre(R292A) and Cre(R173A, R292A) reactions with the joining substrate, we did not detect an accumulation of the MeP–tyrosyl intermediate that is pre- dicted for its ineﬃcient conversion to the hydrolysis or joining products. In all three cases, the cleaved intermedi- ate reached a maximum between 4 and 6% (Figure 8A). Its depletion in the Cre(R173A) and Cre(R292A) reactions was in step with the increase in the joined product. Furthermore, neither strand cleavage nor hydrolysis was improved appreciably when the bottom strand in the joining substrate was phosphorylated at the 50-end (Figure 8B; data not shown). It is the long tail (perhaps in its folded form), rather than the free 50-hydroxyl group, that likely contributes to the inhibition of strand cleavage. This non-standard conﬁguration of the half-site could impede its self-association into dimeric, trimeric and tetrameric assemblies. The consequent adverse eﬀect on the allosteric Figure 8. Strand cleavage and strand joining by Cre(R173A), Cre(R292A) and Cre(R173A, R292A). The MeP-half-sites contained either a 50-hydroxyl (A) or a 50-phosphate group (B) on the bottom strand. The plots in A were obtained by using the data presented in Figure 7. In the reactions shown in (B), no strand joining was possible because of the blocked 50-end of the bottom strand. Global ﬁts to the kinetic data were generated using KinTek Global Kinetic Explorer. Points that fall below the ﬂat portions of the simulated curves in (B) may be due to slow breakdown of the tyrosyl intermediate by a weak type I endonuclease activity, which was not assayed in this analysis.