Fig. 3.

Extension of a ³²P-labeled 25-mer primer opposite next base from G and AP site by human Y-family and B-family DNA polymerases. Primer (25-mer) containing an A, T, G, or C at the 3’end was annealed with each of the two different 36-mer templates (Table 1) containing an unmodified G or AP site (tetrahydrofuran) placed at the 25th position from the 3′-end (see Fig. 1). Reactions were done for 10 min with each of (a) Y-family DNA polymerases: pols η (1 nM), ι (2 nM), κ (10 nM), or REV1 (40 nM) and (b) B-family DNA polymerases: pols α (8 nM) or δ (2 nM)/PCNA (300 nM), with a constant concentration of DNA substrate (100 nM primer/template) as indicated. ³²P-labeled 25-mer primer was extended in the presence of all four dNTPs. The reaction products were analyzed by denaturing gel electrophoresis with subsequent phosphoimaging analysis.