Site-Specific Recombination Involves Breakage and Reunion

KEY CONCEPT
- Cleavages staggered by 7 bp are made in both attB and attP, and the ends are joined crosswise.
The att sites have distinct sequence requirements, and attP is much larger than attB. The function of attP requires a stretch of 240 bp, whereas the function of attB can be exercised by the 23-bp fragment extending from −11 to +11, in which there are only 4 bp on either side of the core. The disparity in their sizes suggests that attP and attB play different roles in the recombination, with attP providing additional information necessary to distinguish it from attB.
Does the reaction proceed by a concerted mechanism in which the strands in attP and attB are cut simultaneously and exchanged? Or,are the strands exc hanged one pair at a time, with the first exchange generating a Holliday junction and the second cycle of nicking and ligation occurring to release the structure? The alternatives are depicted in FIGURE 1.

FIGURE 1. Does recombination between attP and attB proceed by sequential exchange or concerted cutting?
The recombination reaction has been halted at intermediate stages by the use of “suicide substrates,” in which the core sequence is nicked. The presence of the nick interferes with the recombination process. This makes it possible to identify molecules in which recombination has commenced but has not been completed. The structures of these intermediates suggest that exchanges of single strands take place sequentially.
The model illustrated in FIGURE 2. shows that if attP and attB sites each suffer the same staggered cleavage, complementary single-stranded ends could be available for crosswise hybridization.
The distance between the lambda crossover points is 7 bp, and the reaction generates 3′–phosphate and 5′–OH ends. The reaction is shown for simplicity as generating overlapping single-stranded ends that anneal, but actually occurs by a process akin to the recombination event . The corresponding strands on each duplex are cut at the same position, the free 3′ ends
exchange between duplexes, the branch migrates for a distance of 7 bp along the region of homology, and then the structure is resolved by cutting the other pair of corresponding strands.

FIGURE 2. Staggered cleavages in the common core sequence of attP and attB allow crosswise reunion to generate reciprocal recombinant junctions.