FIGURE 8.

DprA antagonizes the inhibitory effect of RecU on DNA strand exchange. Top left, scheme of reactions performed on the left part of the gel (lanes 2–9). The css (+ strand in black) and the homologous lds (– in gray) substrates were preincubated with SsbA, DprA, RecU, and ATP (5 min, 37°C), after which RecA was added. Top right, scheme of the reactions performed on the right part of the gel (lanes 11–18). Here, DNA substrates were preincubated with SsbA, DprA, RecA, and ATP (5 min, 37°C), followed by RecU. The predicted intermediates (jm) and final products (nc) are illustrated. Homologous ssDNA (10 μM) and dsDNA (20 μM) were preincubated with SsbA, DprA and with variable concentrations of RecU (lanes 3–9; doubling from 6.2 to 400 nM) or a fixed RecA concentration (lanes 11–18) in buffer B containing 5 mM ATP (5 min, 37°C). A fixed RecA concentration (lanes 2–9) or variable amounts of RecU (lanes 12–18) were then added and the reaction was incubated (60 min, 37°C). Lane 1, DNA substrate controls (C); lanes 2 and 11, RecU was omitted, and in lane 10, the reaction was terminated after preincubation (5 min) without RecU. Reactions were resolved after deproteinization by 0.8% agarose gel electrophoresis. Band positions for css, lds, cds, jm, and nc are indicated. Bottom, recombination intermediates (jm) and products (nc) are expressed as the percentage in respect to the total substrate added. Results are shown as the mean ± 5% SEM of ≥3 independent experiments.