6600 Nucleic Acids Research, 2010, Vol. 38, No. 19 the topoisomerase mutant lacking Arg-223 (equivalent to Arg-292 in Cre and Arg-308 in Flp), both of which exhibit the type I endonuclease activity towards the MeP–tyrosyl intermediate (9,10). However, the dramatic diﬀerence in stabilities between the P–tyrosyl intermediate and MeP– tyrosyl intermediate seen in topoisomerase or topoisomer- ase mutant reactions is not observed in Cre or Cre mutant reactions. Unlike the toposiomerase situation, the role of phosphate electrostatics per se in protecting the cleaved intermediate against hydrolysis during strand joining by Cre appears not to be prominent. This protection is likely derived from the compact organization of the Cre tetramer in the recombination synapse plus the alignment of the 50-hydroxyl groups in their joining-competent orien- tation concomitant with cleavage. Arg-173 is more important than Arg-292 in strand joining of the MeP–tyrosyl intermediate: recombination in MeP–DNA by Cre(R292A) The sum of the strand cleavage and joining results from the MeP-half-site signify the non-equivalent roles of Arg-173 and Arg-292 in recombination chemistry. Whereas strand cleavage proceeds in the absence of both arginines (7) (Figures 2A and 5A); detectable strand joining requires at least one of the two to be present (Figure 7). Relative cleavage and joining eﬃciencies of Cre(R173A) and Cre(R292A) suggest that Arg-173 is more important in joining than Arg-292. In Flp, the situ- ation is quite diﬀerent. As shown previously (8), replace- ment of Arg-308 in Flp (counterpart of Arg-292 in Cre) blocks strand cleavage by the catalytic tyrosine, causing abortive hydrolysis of MeP. Since little or no tyrosyl inter- mediate is formed by Flp(R308A), strand joining by this mutant has to be correspondingly inept or absent. By contrast, we ﬁnd that Flp(R191A), lacking the equivalent of Arg-173 in Cre, can mediate strand cleavage as well as strand joining (11). Consistent with their abilities to promote both these reactions, Cre(R173A), Cre(R292A) and Flp(R191A) are functional recombinases towards MeP DNA substrates (Figure 9) (11). Stereochemistry of strand cleavage and strand joining in the MeP-half-site reactions In the crystal structure (Figure 1), Arg-173 and Arg-292 are favorably located to make potential contacts with the pro-RP and pro-SP oxygen atoms, respectively, of the scissile phosphate in the intact DNA backbone. When DNA is cleaved by Tyr-324, Arg-173 position is modulated to be able to contact either of these two oxygen atoms. Consistent with the structural data, our previous work (7) revealed a strong preference by Cre(R292A) for the SP form of the MeP-half-site (in which the pro-RP oxygen is replaced by the methyl group and the pro-SP oxygen is retained) over the RP form. The simple expectation is that the preference would be switched to the RP form for Cre(R173A), at least during the cleavage reaction. Because of the modu- lation of phosphate contacts by Arg-173 following cleavage, potential stereochemical bias of Cre(R173A) in the type I hydrolysis reaction or the joining reaction is diﬃcult to anticipate. A more incisive stereochemical analysis of the Arg-173/Arg-292 single and double mutants of Cre using pure RP and SP forms of the MeP substrate is being performed. Substrate and active site electrostatics in DNA phosphoryl transfer: more than just mechanistic? The ﬁndings from this study, along with those previously published (7–10), highlight the signiﬁcance of electrostatic complementarity between active sites and substrates during the phosphoryl transfer steps of site-speciﬁc recom- bination by tyrosine recombinases and DNA relaxation by a type IB topoisomerase. Adverse catalytic eﬀects of mu- tations that eliminate critical positive charges within the active site can be ameliorated by removing a negative charge from the substrate by P to MeP substitution. The extent of suppression is variable depending on the particu- lar active site mutation as well as the particular chemical step of the reaction, stand cleavage or joining. Cre and Flp recombinase mutants that are rescued for both chemical steps (albeit to diﬀerent extents) are also able to perform recombination reaction in MeP–DNA substrates (this study) (11). Chemical modiﬁcation of the phosphate by methyl replacement of non-bridging oxygen and muta- tional alterations of active site residues are not seriously detrimental to the conformational dynamics required for strand exchange between two DNA partners. Phosphate and active site electrostatics, critical as they are in phosphoryl transfer chemistry, may transcend purely catalytic roles. In toposiomerase reaction, the MeP–tyrosyl intermediate is prone to rapid hydrolysis whereas its P–tyrosyl counterpart is extremely resistant to this futile reaction (9,10). In Flp reaction, the absence of the active site Arg-308 makes the MeP bond the target of direct hydrolysis, thus aborting recombination at the cleavage step. This activity, in a much weaker form, has also been observed in a wild-type Flp/MeP-half-site reaction (11) whereas direct hydrolysis of the phospho- diester bond by Flp has been undetectable or insigniﬁcant under normal reaction conditions. Manifestation of such aberrant reactions as a result of phosphate modiﬁcation with or without catalytic mutations suggests that substrate and active site electrostatics may be utilized for protecting desired reactions against wanton side reactions that damage genome integrity. Marked instability by hydrolysis of either the DNA backbone or that of the tyrosyl intermediate by MeP sub- stitution in conjunction with removal of catalytic arginines has not been observed in Cre (this study) (7). Features other than electrostatics such as active site organization, tightness of the DNA–protein reaction complex and the conformational dynamics of DNA cleavage and joining may also serve to exclude water. Given that a wide variety of biologically critical transesteriﬁcation reactions (DNA recombination, DNA transposition and RNA splicing, to name a few) must occur in the context of an overwhelming excess of water ( 56M), strategies to minimize hydrolysis must be an inherent part of the evo- lutionary designs of active sites performing these reactions.