Figure 4.

Time-dependent product accumulation in reactions with different enzyme concentrations. (A) Different amounts of purified ROS1 (2.25 nM, filled circles; 4.5 nM, open circles; 22.5 nM, filled triangles), were incubated at 30°C with a double-stranded oligonucleotide substrate (20 nM) containing a 5-meC:G pair. (B and C) Reaction was started by addition of ROS1 (4.5 nM) and additional enzyme aliquots (4.5 nM) were added at 4 and 8 h (arrows). Reactions were stopped at the indicated times, products were separated in a 12% denaturing polyacrylamide gel (C), and the amount of incised oligonucleotide was quantified by fluorescence scanning (B).