Figure 3. Extension of a ³²P-labeled primer across G or N²-AnthG by human wild-type and variant pol κ (1–526) enzymes.

Two different 36-mer templates containing an unmodified G or N²-AnthG was annealed with primer (18-mer), to make primer-template DNA substrates positioning the 3′-end of primers just before a G or N²-AnthG in the template strand. Reactions were done for 15 min with increasing concentrations of pol κ (1–526) (0 – 1.5 nM) and DNA substrate (100 nM primer/template) as indicated. ³²P-labeled primer was extended in the presence of all four dNTPs (25 µM each). The reaction products were analyzed by denaturing gel electrophoresis and phosphorimaging. (a) Extension across G. (b) Extension across N²-AnthG.