6598 Nucleic Acids Research, 2010, Vol. 38, No. 19 interaction between two neighboring Cre monomers is expected to be detrimental to strand cleavage. Strand cleavage in full-sites has been shown to be stimulated strongly by synapsis between two partner sites (20). However, despite the unfavorable eﬀect of the special half-site design on strand cleavage, the data in Figure 7 demonstrate the distinct joining eﬃciencies of wild-type Cre in the P-half-site and Cre(R173A), Cre(R292A) and Cre(R173A, R292A) in the corresponding MeP-half-site. The more or less equivalent yields of the MeP–tyrosyl intermediate from the strand joining substrate by the single and double mutants (Figure 8A), contrast with the distinct outputs of the joined product by them (Figure 7). The 4-fold better joining by Cre(R292A) than Cre(R173A), with no detectable joining by Cre(R173A, R292A), suggests that Arg-173 is more critical than Arg-292 for strand joining in the MeP-half-site. Recombination between MeP-full-site partners by Cre(R292A) The ability of Cre(R292A) to promote strand cleavage and strand joining (although with reduced eﬃciency) in MeP-half-sites suggests that this mutant may promote re- combination between two full-site partners containing MeP at the scissile positions. A successful reaction would indicate that neither MeP substitution nor the lack of this catalytic arginine poses a strong barrier to the conformational dynamics of strand exchange between DNA partners. Cre(R173A) may also perform recombination in MeP-full-sites, perhaps with lower eﬃ- ciency because of its weaker joining capacity. In the MeP-full-site substrates, because of the nature of their construction (see ‘Materials and Methods’ section), the recombination target sites were rendered palindromic in sequence. The symmetrized strand exchange region (50ATGT-ACAT30) would not aﬀect recombination (22), although the reaction would lose the directionality associated with asymmetric strand exchange regions, as dictated by the requirement of homology between them. One of the reaction partners was lengthened at either end by polymerizing an additional oligonucleotide tail. DNA exchange between the radioactively labeled short substrate and the unlabeled long one would result in a readily iden- tiﬁable recombinant strand (Figure 9). In the MeP-full-site reaction, Cre(R292A) generated the predicted 74-mer (Figure 9A) or the 70-mer (Figure 9B) product of strand exchange. The size diﬀerence was due to the 4-nt shorter tail length of the unlableled substrate (S2) in the reaction shown in Figure 9B. The 70-mer yield of 5% in 4–6 h is in line with the 3% yield of the joined product obtained in the same time period during the joining reaction in the MeP-half-site (Figure 7B). Cre(R173A) also carried out recombination in the MeP-full-site, its activity being weaker than that of Cre(R292A) (Figure 9C). This result is consistent with the poor joining eﬃciency of Cre(R173A). For compari- son, recombination between two P-full-sites by Cre is shown in Figure 9D. Thus, Cre(R173A) and Cre(R292A) are weak but func- tional tyrosine recombinases for DNA substrates in which the negative charge at the scissile positions is neutralized by MeP modiﬁcation. The data also indicate that neither the lack of Arg-173 or Arg-292 in the Cre active site nor the MeP substitution in DNA precludes the conform- ational dynamics associated with the strand exchange process. DISCUSSION In this study, we have addressed the role of electrostatic complementarity between the Cre recombinase active site and the scissile phosphate in the strand cleavage and joining steps of recombination. Substrate and active site electrostatics were modulated by modifying the scissile phosphate to methylphosphonate and replacing speciﬁc arginine residues with alanine, respectively. As deduced from crystal structures, interactions between the phos- phate and each of the two conserved arginines, Arg-173 and Arg-292, of the catalytic pentad provide the major electrostatic contribution towards stabilizing the transi- tion state (2,3). The catalytic properties of active sites lacking either one or both of these positively charged side chains towards the charge-neutralized scissile MeP reveal mechanistic features of recombination that could not have been gleaned from reactions with DNA sub- strates containing native phosphate groups at the scissile positions. The cumulative results from this study are summarized in Table 2, and provide the framework for active site mechanisms considered below. Dispensability of Arg-173 and Arg-292 in strand cleavage by Tyr-324 in MeP–DNA The present work, in conjunction with previous ﬁndings (7), demonstrates that Cre variants lacking either or both of the conserved active site arginines (Arg-173 and Arg-292) are capable of MeP activation and cleavage using Tyr-324 nucleophile (Figures 2A and 5A). Cleavage competence in the combined absence of the conserved catalytic arginines was not anticipated from structural data or previous biochemical results with Cre mutants on substrates containing scissile phosphates. When the nominal negative charge in the transition state is reduced from 2 to 1 by P to MeP substitution, an active site with severe electrostatic impairment is still able to promote the ﬁrst step of recombination with reasonable eﬃciency. The MeP transition state with its lone negative charge could be stabilized through electrophilic or other types of interactions from the intact pentad residues (Lys-201, His-289 and Trp-315), perhaps with additional assistance from non-pentad residues. According to the crystal structure, both His-289 and Trp-315 are within hydrogen bonding distance in the active site containing the P–tyrosyl intermediate, and Trp-315 is also in contact with the scissile phosphate when it is not cleaved (2). Furthermore, the active site cavity resulting from the absence of Arg-173 and/or Arg-292 may permit residues that are normally non-catalytic to relocate themselves. For example, if Gln-133 or Arg-326 could swing into favorable positions, they might serve a surrogate role in transition state stabilization by the mutant active sites.