FIGURE 7.

Anti-OsXRCC1 serum inhibits 3′-phosphate end processing and ligation of DNA demethylation intermediates catalyzed by cell extracts. A, analysis of DNA incision products generated on the upper strand during DNA demethylation. Purified ROS1 (70 nm) was incubated at 30 °C for 8 h with a 51-mer double-stranded oligonucleotide substrate (20 nm) containing a single 5-meC:G pair. Reaction products, which contained a mixture of β- and β,δ-elimination products (lane 1), 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, 5, and 7 μl) and all four dNTPs, except when indicated. Reactions were stopped, and products were separated in a 12% denaturing polyacrylamide gel and detected by fluorescence scanning. The lower panel shows the percentage of 3′-phosphate (3′-P) not converted into 3′-OH in two independent experiments. B, DNA ligation assay. DNA duplexes containing a nick in the upper, 5′-Alexa Fluor-labeled strand were incubated with 35 μg of extract from WT plants at 30 °C for 3 h in a reaction mixture supplemented with increasing amounts of anti-OsXRCC1 serum (0, 2.5, 5, and 7 μl) and all four dNTPs, except when indicated. Reaction products were separated using a 12% denaturing polyacrylamide gel and detected by fluorescence scanning. The lower panel shows the percentage of fully ligated products. Values are mean ± S.E. from two independent experiments.