Figure 5.

Gel retardation assay performed under denaturing conditions to detect the formation of a covalent linkage between oligomeric top- and bottom strands (o-0t, o-0b) of the wild-type attC and integrase. Protein fractions and concentrations as well as incubation times are indicated. The oligonucleotides were labelled with ³²P and used at 1–6 nM, corresponding to ∼200–300 c.p.s. per binding mixture. The mutation in IntI1, Q255P, is abbreviated with an asterisk. For control experiments and more details see Materials and Methods. (i) A comparison of the binding of IntI1^* and of IntI1 to top- and bottom strands of wild type attC in the presence of 0.42 μg poly(dI–dC)•poly(dI–dC). (ii) Assay at different incubation times before addition of SDS stop solution. (iii) Assay where the bottom strand of wild-type attC was mixed with IntI1^* or IntI1^*Y312F, in which the nucleophilic tyrosine is substituted with a phenylalanine. B, protein–DNA complexes; F, free DNA.