Figure 5.

Restriction enzyme protection assay for homologous pairing between linear or circular RecA–ssDNA with relaxed, supercoiled or linear duplex DNA with 120 bp of homology. (A) (Top) An agarose gel that shows homologous pairing between a 520mer circular RecA–ssDNA (C-520, with 120 nt homologous to the duplex) filament and relaxed circular duplex target. (Upper middle) A reaction in which supercoiled duplex DNA was used as the target; (lower middle) the same reaction with linear duplex DNA as the target. (Bottom) Quantification of the three reactions (diamonds represent the reaction with relaxed duplex target; squares represent the reaction with supercoiled duplex target; triangles represent the reaction with linear duplex target). The reaction times were 5, 10, 20, 30, 40 and 60 min after formation of synaptic complexes and prior to 8 min of restriction digestion (see Materials and Methods for details). (B) Here, instead of 520mer circular RecA–ssDNA a 120mer homologous linear RecA–ssDNA (H-120) was used as the searching molecule. Top to bottom the panels show the formation of synaptic complexes with all three types of target duplex DNA as in (A). Solid lines indicate homology between the two molecules. Arrows to the right of each gel indicate the initial substrate used in that reaction.