Figure 1.
(A) Topology of “random-collision” site-specific recombination. The diagram shows a planar projection of a prototypical supercoiled DNA substrate undergoing intramolecular site-specific recombination. Recombination sites, indicated by arrowheads, divide the DNA contour into two distinct domains, shown as shaded and outlined regions. Relative motion of these sites along the superhelix axis is termed slithering (first column). This motion generates a variable number of interdomainal superhelical turns, which are trapped at site synapsis in the folded conformations (second column). Recombination (third column) and subsequent nicking of the DNA to remove residual supercoiling (fourth column) generates knotted products in which the signed number of irreducible knot crossings (Kn, given below and to the right of each knot) is proportional to the number of interdomainal supercoils trapped at synapsis. Additional supercoiling-independent crossings appear in each product as a consequence of the mechanism of recombination. The presence of a bent or flexible insert, indicated by the solid segment, can bias the distribution of knotted products by localizing in the terminal loops of the superhelix, as in the case of the conformation shown at the bottom. With the symmetrical arrangement of recombination sites shown, the most favorable configurations correspond to juxtaposition of the recombination sites across the superhelix axis, which biases the distribution of recombination products in favor of simple topologies. (B) Plasmid DNAs used in these studies. Recombination substrates were derived from a plasmid designated patt4.5i, which positions the centers of the att sites relative to the SmaI (S) cloning site as indicated. A-tract-containing plasmids, pA5N5i and pA5N1i, and control plasmid pRani were constructed by cloning pentameric repeats of the 21-bp double-stranded oligonucleotide sequences shown into the SmaI site of patt4.5i. The 105-bp insertions are indicated by the solid segment in the plasmid map. (C) Electrophoresis in an 8% polyacrylamide gel of 138-bp KpnI–HindIII fragments containing synthetic inserts. Lanes: L, 100-bp ladder; 1, [A5N1]5; 2, [A5N5]5; 3, [Ran]5.