Figure 3.

Strategy to determine which dNMP is recovered opposite the template lesion from primer extended by pol δ. After annealing of 5′-³²P-labeled primer to a template containing an abasic site at position X (I), the template-primer was incubated with proteins and products purified by standard denaturing PAGE. Reaction products (II) extended by only a single nucleotide could be analyzed directly. Fully and near-fully extended products were reannealed to the template on which they were synthesized and the resulting oligonucleotide was treated with EcoRI restriction endonuclease to generate the ³²P-labeled product shown (III). After two-phase PAGE, each of the oligonucleotides shown (IV) could be separately resolved; hence with appropriate mobility standards, it is possible to determine the nucleotide sequence of the reaction product (53).