Figure 6
Effect of various purified PCNA fractions to promote nucleotide incorporation by calf thymus pol δ beyond chemically defined template base lesions. A: the structure of the 5'-32P-labeled 30-21-mer template-primer; only the primer (21-mer) was radiolabeled and X indicates the position of a modified tetrahydrofuran moiety (model abasic site) on the 30-mer template. B: lane 1, gel-purified primer alone was subjected to electrophoresis; lanes 2–6, incubations were formulated as indicated with the template-primer shown in A followed by standard denaturing PAGE. h, human; dr, Drosophila melanogaster. For lanes 2–6, each incubation contained 0.5 pmol of labeled primer (3'-OH ends) annealed to 0.5 pmol of template (3'-OH ends), 10 ng pol δ and 70 ng PCNA as indicated. NH2-terminally his-tagged-PCNA fractions are as indicated; wt, wild-type; Q123V, recombinant Drosophila PCNA containing a single amino acid, glutamine at position 123, changed to valine; dr119-133h, recombinant Drosophila PCNA containing the entire interdomain connector loop (amino acids 119-133) replaced with the corresponding human PCNA amino acids.