Figure 6.

RecA·dATP-mediated recombination in the absence or presence of SsbA. (A) Circular 3199 nt ssDNA (10 μM) and the 3199 bp KnpI (In lanes 1–4) or EcoRI-linearised duplex (lanes 5–7) or the 4374 bp EcoRI- (Sub A, lanes 8–11), PstI- (Sub B, 12–14), or NcoI–linearized duplex (lanes 15–17) substrate (20 μM in nt) was incubated with RecA (0.8, 1.0 and 1.2 μM) in buffer A containing 5 mM dATP for 60 min at 37°C, and separated by 0.8% AGE. (B) Circular ssDNA and duplex substrate of part (A) were pre-incubated with SsbA (0.3 μM) in buffer A containing 5 mM dATP for 5 min at 37°C. RecA (0.8, 1.0, 1.2 and 1.4 μM) was added, the reaction was incubated for 60 min at 37ºC and then separated by 0.8% AGE. In lanes 2-5 the 3199 bp KnpI–linearized duplex DNA was used. The symbols + and – denote the presence or absence of the indicated protein(s). The positions of the bands corresponding to css, lds, cds, prd and jm are indicated. A cartoon of the lds substrate with homology (in black) and heterology (in red) with the css substrate are indicated, with the broken line at the centre of each condition. The results are the average of more than three independent experiments (the results given are within a 5% standard error).