FIGURE 2.

Substitution of Arg-363 with alanine effects dATP, dCTP, and dGTP usage in unwinding DNA. DNA unwinding activity of wild-type helicase (A) and helicase-R363A (B). Reactions for measuring the unwinding of DNA were carried out as described under “Experimental Procedures.” The reaction contained a 5′-³²P-labeled 75-mer oligonucleotide annealed to a 95-mer oligonucleotide (see the inset), a 5 mm concentration of the indicated dNTP, and 100 nm helicase. After incubation at 37 °C, aliquots of the reaction were removed at the indicated time of incubation. The products were then separated on 10% non-denaturing polyacrylamide gels in 1× Tris borate-EDTA buffer. The amount of labeled single-stranded oligonucleotide separated from the duplex DNA substrate in each case was calculated and expressed as percent of total helicase substrate. Results are the average of three experiments.