6596 Nucleic Acids Research, 2010, Vol. 38, No. 19 Figure 6. Lack of endonucleolytic activity in MeP-half-site reactions of Cre(R173A, R292A, Y324G). Reactions and product analysis were per- formed as described under Figure 2. The predicted positions of migration of the covalent intermediate and the hydrolysis product (HP) are indicated. The incubation time for the control reaction without addition of Cre(R173A, R192A, Y324G) was 24 h. (A) SDS-PAGE. (B) Urea-PAGE. Figure 7. Strand joining reactions of Cre(R173A) (A), Cre(R292A) (B) and Cre(R173A, R292A) (C) in an MeP- half-site and Cre (D) in a P-half- site. In the schematic diagram, the scissile MeP or P position is indicated by the ﬁlled circle. The bottom strand of the half-site designed to follow strand joining was longer than that of the standard MeP-half-site used in the strand cleavage/hydrolysis assays, and contained a free 50-hydroxyl group. Looping back of the single-stranded region would place the 50-hydroxyl in position to attack the tyrosyl intermediate formed by strand cleavage. This intra-half-site reaction would produce a hairpin (63-mer). Strand joining could potentially occur between two half-sites to yield a ‘pseudo-full-site’. The individual strands of the pseudo-full-site and the hairpin would be identical in sequence, and display the same mobility during denaturing PAGE. The lower panels show urea–PAGE analysis to assay type I endonuclease activity. ‘JP’ refers to this joined product, ‘HP’ to the hydrolysis product and ‘S’ to the labeled strand of the half-site. ‘C’ is a control reaction incubated for 24 h in the absence of Cre or a Cre variant. raise questions of their potential catalytic competence in strand joining as well as their eﬀects on the competition between the joining reaction and the type I endonuclease activity, both of which target the tyrosyl intermediate. To test joining, Cre(R173A), Cre(R292A) and Cre(R173A, R292A) were reacted with an MeP-half-site containing a longer 50 single-stranded extension of the bottom strand (with a hydroxyl end) than the standard MeP-half-site used in strand cleavage reactions. This tail could loop back and pair with itself to position the 50-hydroxyl in the joining orientation (Figure 7). Cre(R173A) yielded 1.5% of the joined product (Figure 7A), whereas Cre(R292A) was more eﬃcient, the output leveling oﬀ at 6% (Figure 7B). Cre(R173A,