Figure 1.

Substrates, products, and complexes in L5 integrative recombination. (A) Scheme for recombination in vitro with linear substrates. Linear attP DNA radiolabeled on either one or both ends is incubated with a 45-bp attB DNA, Int-L5, and mIHF. Recombination of the substrates yields two products of intermediate sizes, attR and attL. Arm-type sites required for integration are in black, and the dispensable P3 site is in white (the dispensable P6/P7 pair of sites to the right of P4/P5 is not shown), with relative directionalities indicated by arrows (6). The gray boxes indicate loose inverted repeats at the core that are protected from DNase I by Int-L5 (6). The horizontal bars within attP are protected by mIHF in in situ footprinting of the intasome and SC1 (7). (B) The products of integrative recombination. Reactions containing attP DNA radiolabeled on the P1 end were performed either on ice or at room temperature (rt; as indicated) as described in Materials and Methods and loaded onto a native polyacrylamide gel. The positions of complexes, attR product, free attP, and intasome (intsm), and the origin of electrophoresis (O) are indicated. (C) Synaptic complex 2 (SC2) is mIHF-independent and contains one attB molecule. Complexes were formed on ice as described for B by using linear attB DNAs of 45 bp, 126 bp, or both (as indicated). The positions of SC1 as formed with the 45-bp attB and of SC2 as formed with either the 45-bp or 126-bp attB are indicated. SC1 formed with the 126-bp fragment migrates slowly and is not separated from the loading wells. Because only two SC2 bands are seen with two differently sized attBs, SC2 contains attP and only one molecule of attB. Note that the mobilities of the complexes also vary with the size of attP DNA used (compare with B).