more eﬃcient than Cre(R173A); the double mutant Cre(R173A, R292A) does not give detectable joining. Furthermore, Cre(R173A) and Cre(R292A) carry out re- combination between two MeP–DNA partners. Thus, speciﬁc catalytic deﬁciencies resulting from the loss of one or both conserved arginines of the Cre catalytic pentad can be suppressed to varying extents by neutralizing the negative charge on the scissile phosphate. We consider the mechanistic implications of these ﬁndings from the general perspective of phosphoryl transfer reac- tions in nucleic acids. MATERIALS AND METHODS Synthetic oligonucleotides and assembly of half-site substrates Standard oligonucleotides were purchased from IDT (Coralville, IA). The methylphosphonate containing oligonucleotide (a racemic mixture of the RP and SP forms) was supplied by Trilink Technologies (San Diego, CA). Hybridization of oligonucleotides was performed as described previously (16). A list of the oligonulceotides used in this study is summarized in Table 1. Construction of P- and MeP-full-sites for recombination reactions The short P-full-site recombination partner was assembled from two 54-mer synthetic oligonucleotides (7), and the long one by PCR ampliﬁcation of a DNA segment con- taining LoxP using appropriate primers. To construct the MeP-full-site substrates for recombin- ation, the 27-mer oligonucleotide that served as the scissile strand in the MeP half-site was extended by Klenow poly- merase (exonuclease minus) using a 45-mer synthetic template strand carrying a 30 dideoxy-C end. The 54-mer product strand so formed was puriﬁed away from the template by urea–polyacrylamide gel electrophoresis (urea–PAGE) and DNA isolation. This DNA strand Nucleic Acids Research, 2010, Vol. 38, No. 19 6591 could hybridize to itself to generate the short MeP–LoxP recombination partner containing a symmetric strand exchange region. To obtain the long MeP–LoxP partner, the MeP containing 27-mer was lengthened at its 30-end to a 70-mer by the Klenow polymerase chain elongation method using a 61-mer template strand with a 30 dideoxy-C end. The design of the template strand was such that the polymerized product contained the 54-mer sequence of the short MeP–LoxP plus 16 nt as a 30 ap- pendage. Self-annealing of the puriﬁed 70-mer yielded a variant of the 54-mer palindromic full-site carrying 16-nt long single-stranded 30 tails on both strands. Note that the blocked 30-ends (dideoxy-C) of the template strands kept them from being extended during the Klenow polymerase reaction. A second long MeP-full-site was constructed by a splint-oligo assisted ligation of the 27-mer MeP containing strand to an all phosphate containing 47-mer oligonucleo- tide, followed by gel puriﬁcation and self-annealing of the 74-mer. The diﬀerence between the two versions of the full-sites was only in the length of the single-stranded tails, 20 versus 16 nt. Puriﬁcation of Cre and Cre mutants Wild-type Cre and its mutant derivatives, harboring a His-6 tag at their N-termini, were puriﬁed as described previously (7,17). In vitro Cre reactions In vitro Cre reactions were carried out under conditions described earlier (7). Assays were initially optimized using a range of Cre and Cre variant amounts per Cre binding element of the DNA substrate employed. Subsequent ex- periments were performed with the optimized protein amounts. Reactions were terminated by addition of sodium dodecyl sulfate (SDS) to a ﬁnal concentration of 0.1%. For assaying the Cre–DNA covalent adduct, samples were analyzed directly by SDS polyacrylamide Table 1. Synthetic oligonucleotides used in the assembly of half-site and full-site substrates for Cre reactions are listed Oligomer MeP-half-site (top strand) P-half-site (top strand) P- or MeP-half-site (bottom strand); hydrolysis assays P- or MeP-half-site (bottom strand); strand joining assays Template oligo used to synthesize the 54-mer strand (top and bottom) of the short MeP-full-site by Klenow polymerase ﬁll-in reaction Template oligo used to synthesize the 70-mer strand (top and bottom) of the long MeP-full-site by Klenow polymerase ﬁll-in reaction 47-mer used to generate a 74-mer strand (top and bottom) of a long MeP-full-site by ligation to the 27-mer shown in row1 splint oligo used in the ligation to generate the 74-mer strand of the long MeP-full-site Size (bases) Sequence 27 27 30 39 45 61 47 44 50 ACTTGGATCCATAACTTCGTATAA(mp)TGT 30 50 ACTTGGATCCATAACTTCGTATAATGT 30 50 CATACATTATACGAAGTTATGGATCCAAGT 30 50 TGTATGTTTCATACATTATACGAAGTTATGGATCC AAGT 30 50 ACTTGGATCCATAACTTCGTATAATGTACATTATAC GAAGTTAT(ddC) 30 50 GCATGCATGCATGCATACTTGGATCCATAACTTCGT ATAATGTACATTATACGAAGTTAT(ddC) 30 50 ACATTATACGAAGTTATGGATCCAAGTATGCATGCA TGCATGCATGC 30 50 GATCCATAACTTCGTATAATGTACATTATACGAAGT TATGGATC 30 The string of nucleotides shown in bold represents the Cre binding element. The scissile phosphate or methylphosphonate is indicated by ‘p’ or ‘mp’, respectively. The template oligos employed in the Klenow polymerase reactions contained a 30-terminal dideoxy-C (ddC).