FIGURE 3.

Characterization of Xenopus TDG protein. A, native TDG from E. coli was purified and analyzed by SDS-PAGE and Coomassie Blue staining. B, schematic of the base release assay used to monitor TDG activity. Double-stranded DNA (29 bp) containing a site-specific G-T mispair is 5′-radiolabeled on the DNA strand containing the thymine of the mispair. TDG activity generates an abasic site, and subsequent treatment with sodium hydroxide and heat leads to hydrolysis of the backbone at the abasic site. TDG activity is measured as the appearance of a 14-nt product. As a control, the oligonucleotide was treated with EcoRI, which cleaves adjacent to the G-T mispair in the fully complementary duplex to generate a 12-nt product. C, recombinant wild type TDG and all TDG mutants used in this work, except the catalytically inactive form (CI), are active in the base release assay diagramed in B. Detailed descriptions of these mutants can be found upon their first experimental usage in Figs. 5 and 6. The concentration of TDG in all reactions was 100 nm, and the DNA concentration was 50 nm. Strand cleavage was measured using a 15% polyacrylamide gel.