FIGURE 12.

The A11V mutation improves RecA filament dynamics relative to filaments made with RecA K250R. Reactions were carried out as described under “Experimental Procedures” and contained 4 μm M13mp18 circular ssDNA, 4 μm M13mp18 linear duplex DNA (where indicated), 2.5 μm RecA protein or RecA protein mutant, 0.4 μm SSB protein, and 3 mm ATP. A-C show reactions carried out by wild-type (WT) RecA protein, RecA K250R protein, and RecA K250R/A11V protein, respectively. ATP hydrolysis is monitored spectrophotometrically, as described under “Experimental Procedures.” Three reactions are shown for each protein. The linear duplex DNA was added to two reactions at the time indicated, illustrating the decline in ATP hydrolysis that generally accompanies the initiation of DNA strand exchange. One of these two reactions was further challenged by the addition of 5 μm RecA K72R protein at the time indicated. In each panel, the top line (labeled NA for no addition) represents a reaction that contains only ssDNA and the indicated RecA protein, with no dsDNA or RecAK72R additions. lds, linear dsDNA reactant.