6590 Nucleic Acids Research, 2010, Vol. 38, No. 19 Figure 1. Organization of the catalytic pentad and the tyrosine nucleo- phile in the Cre active site. The arrangement of the catalytic pentad residues (R-173, K-201, H-289, R-292 and W-315) and the tyrosine nucleophile (Y-324) in the uncleaved (top) and cleaved (bottom) states of the scissile phosphate are shown. Note that Arg-173 and Arg-292 make hydrogen bonding contacts with the phosphate in both states, with slight changes in their relative positioning. The structural representations are based on the Protein Data Bank ﬁle 1CRX (2), and is represented using the PyMOL molecular graphics system (DeLano Scientiﬁc). Potential hydrogen bond contacts were derived using RasTop v2.6 by imposing a distance constraint of 3.5 A˚ or less. endonuclease activity that directly targets the MeP bond for hydrolysis (see below). The type II activity has been detected in a mutant of the Cre related-Flp recombinase containing alanine in place of Arg-308 (8), which is the counterpart of Arg-292 in Cre. The type IB vaccinia topoisomerase (hereafter referred to as just topoisomerase) or its mutant derivative lacking Arg-223 (which corresponds to Arg-292 in Cre) can also perform strand cleavage in MeP–DNA (9,10). However, the tyrosyl intermediate is highly unstable, being rapidly hydrolyzed to the 30-MeP product. The extreme instability of the MeP–tyrosyl adduct contrasts sharply with the high stability of the P–tyrosyl adduct formed in a wild-type topoisomerase reaction, their respective half-lives at physiological pH being 2 min and 36 days (9). By contrast, the P–tyrosyl intermediate formed by wild-type Cre and the MeP–tyrosyl intermediate formed by Cre(R292A) are hydrolyzed at comparable rates (7). In Flp recombinase, lack of Arg-308 elicits a strikingly distinct response to MeP–DNA from that observed in Cre or topoisomerase. Flp(R308A) utilizes water, rather than the catalytic Tyr-343, as the nucleophile to directly hydro- lyze the MeP bond (8). This futile type II endonuclease activity is also elicited in wild-type Flp by MeP, but to a much smaller extent (11). A unifying feature of Cre, Flp and topoisomerase in their reactions with MeP–DNA is the dispensability of a conserved arginine residue in the MeP activation step. However, as alluded to above, the three also diﬀer in the subsequent chemical outcomes in mechanistically interest- ing ways. Flp, Cre(R292A), topoisomerase and Topo(R223A) mediate strand cleavage in the MeP–sub- strate using the active site tyrosine nucleophile. However, the MeP–tyrosyl intermediates formed in the recombinase and topoisomerase reactions diﬀer signiﬁ- cantly in their relative stabilities with respect to the cor- responding P–tyrosyl intermediates (7–10). Flp(R308A) stands apart in the unusual and strong (stronger than Flp) direct hydrolysis reaction that MeP evokes in it. As explained below, these similarities and diﬀerences can be rationalized by the organization of the active sites of these enzymes and the DNA–protein complexes within which they perform their respective functions. Cre and topoisomerase harbor their active sites entirely within monomers (3,12,13), whereas Flp assembles a shared active site between two neighboring monomers: one donates the pentad, the other the Tyr-343 nucleophile (1,14,15). The coincidence of DNA binding by a monomer and engagement of the scissile phosphate by the tyrosine nucleophile would likely preclude direct hydrolysis of the scissile phosphate by Cre and topoisomerase. The delay between phosphate activation by a DNA-bound Flp monomer and tyrosine delivery by a second such monomer could increase the likelihood of nucleophilic attack by water. The conformational ﬂexibility associated with strand rotation in the DNA relaxation complex might account for the potential susceptibility of the tyrosyl intermediate formed by topoisomerase to hydroly- sis. The compact tetrameric organization of Cre and Flp on the synapsed DNA partners and the conformational dynamics of the cleaved strands within the recombination complex might reduce the risk of hydrolysis of the tyrosyl intermediate formed by the recombinases. It has been sug- gested that Arg-308 positive charge and phosphate negative charge may help exclude water, and prevent abortive hydrolysis, during strand cleavage by Flp and strand joining by topoisomerase, respectively (8–10). Substrate and active site electrostatics may not only con- tribute to the mechanism of phosphoryl transfer but also help avoid untoward side reactions. The present work demonstrates that MeP activation by Cre during strand cleavage takes place in the absence of Arg-173, the other conserved arginine of the Cre active site, or simultaneous absence of both Arg-173 and Arg-292. The single Arg-to-Ala mutants are capable of promoting strand joining as well, Cre(R292A) being