FIGURE 3.

DNA unwinding activity of gp4 with substitutions for Phe⁵²³. A, four different duplex DNAs, each with a different terminus, were examined for their ability to be unwound by wild-type gp4. gp4 unwinds the duplex containing both a single-stranded 3′- and 5′-tail but not those bearing a blunt end, a 5′-tail, or a 3′-tail. The reactions contained 50 nm wild-type gp4 and 100 nm DNA substrates as depicted in the figure. Incubation was at 37 °C for the indicated times and the reactions were carried out as described under “Experimental Procedures.” B, DNA unwinding activity of gp4 variants with Phe⁵²³ substitutions compared with that of wild-type gp4. The DNA substrate contains a replication fork at one end allowing gp4 to assemble on the 5′-single-stranded tail (see inset). The reactions were carried out as described under “Experimental Procedures.” The reactions contained 50 nm gp4, 100 nm DNA, and 1 mm dTTP. After incubation at 37 °C the reaction was stopped at the indicated time points. The unwound ssDNA was separated from the dsDNA substrate in a 10% nondenaturing polyacrylamide gel. The identities of each of the gp4 variants are indicated. The separation of unwound ssDNA from the dsDNA substrate can be seen from the gel picture. The band intensities in each case were measured and plotted in the graph shown in C. C, the percentage of ssDNA unwound from 100 nm substrate by wild-type or altered gp4s was plotted against the time of reaction. Error bars represent the standard deviation of the results from three independent experiments.