FIGURE 6.

Anti-XRCC1 serum inhibits completion of DNA demethylation in cell extracts. A, detection of recombinant Arabidopsis XRCC1 protein (0.5 μg) by Western blotting using serum against OsXRCC1. Recombinant ZDP protein (0.5 μg) was used as a control. B, purified ROS1 (70 nm) was incubated at 30 °C for 8 h with a double-stranded oligonucleotide substrate (20 nm). Reaction products were purified and incubated with 35 μg of cell-free extract from WT plants at 30 °C for 3 h in a reaction mixture that contained increasing amounts of anti-OsXRCC1 serum (0, 2.5, and 5 μl) and either no dNTPs or all four dNTPs, as indicated. Reactions were stopped, and products were digested with HpaII and separated in a 12% denaturing polyacrylamide gel for detection with fluorescence scanning. C, the percentage of fully demethylated products. Values are mean ± S.E. from two independent experiments.