Nucleic Acids Research, 2010, Vol. 38, No. 19 6599 Figure 9. Recombination mediated by Cre(R173A) and Cre(R292A) between LoxP sites containing MeP at the scissile positions. Strand exchange reactions between two MeP-full-sites or two P-full-sites are diagrammed schematically at the top. In the MeP-full-sites, because of their assembly protocol, the strand exchange regions as well as the ﬂanking sequences are fully palindromic, that is, the top and bottom strands are identical in sequence. The single-stranded tails in the long substrate (S2) permit distinction of the recombinant product formed by strand exchange with the end-labeled short substrate (S1) when analyzed by urea–PAGE. The scissile MeP positions are indicated by ﬁlled circles. In (A), S2 to S1 molar ratios were 0.25, 0.5 and 1.0 in lanes 3, 4 and 5, respectively, with a constant reaction time of 3 h; in B and C, S2 to S1 ratio was held at 4:1, and reaction times were varied. (B and C) Depict distinct sections of the same gel, with the control lance (1 in B and 10 in C) being identical. The incubation for the mock reaction without Cre was 4 h. The reaction shown in D utilized P-full-sites (S0 and S00) with the native asymmetric strand exchange regions. In lanes 2–4, the molar ratio of S00 to S0 was maintained as 1:1, with serial 2- and 4-fold increases in the amount of Cre. The reaction time was 1 h. The recombinant products from the MeP- and P-full-site reactions are represented as R and R0, respectively. The single arginine mutants and the double mutant are also capable of hydrolyzing the cleaved intermediate by the type I endonuclease activity (Figures 2B and 5B) (7). These Cre mutants are strikingly diﬀerent from a Flp mutant lacking Arg-308 (equivalent to Arg-292 in Cre), which causes direct hydrolysis of the MeP bond by the type II endonuclease activity (8). This distinction between Cre and Flp appears to follow from their cis- acting monomeric and trans-acting shared active sites, re- spectively. The Cre mutants are akin to topoisomerase and