Fig. 7.

An F52/R54 interface mutation (R54E) inhibits recombination in vitro when site II is present and the IHF concentration is low. Recombination of supercoiled site I^AT, resF^AT and resF^AC substrates (∼8 nM) by the activated mutant N72D/I100T (∼250 nM) and its derivative R54E/N72D/I100T (∼300 nM), in the presence of 0, 0.5 or 3.0 μg ml⁻¹ IHF (0, 23 or 136 nM), as indicated. Reaction samples taken at 9 and 60 min were digested with XhoI and analysed on a 1% agarose gel. The site I^AT and resF^AT/resF^AC substrates give substrate (subs.) and product (resn., resolution; invn., inversion) fragments of different sizes. The resF^AT and resF^AC sites are identical apart from the dinucleotide at the centre of site I; they were referred to previously as resF^E and resF^D respectively (Rowland et al., 2005). Note that the concentration of free IHF will be lower than expected, because IHF binds with high affinity to vector sequences in the supercoiled DNA substrate (data not shown; Prentki et al., 1987).