FIG. 3.

The PIP1 motif of Polι is sufficient to mediate the stimulatory effect of PCNA on the DNA synthetic activity of Polι. The reaction mixtures contained wild-type or mutant Polι proteins (1 nM each), along with singly primed 75-nucleotide-long DNA substrate (10 nM) in which the 29-nucleotide primer was ³²P labeled at its 5′ end, and the template contained biotin-streptavidin complex at both ends to prevent the PCNA sliding off the DNA and all four deoxynucleotides (100 μM each), in the presence or absence of PCNA (50 ng), RFC (5 ng), and RPA (50 ng). After incubation for 10 min at 37°C, samples were quenched and run on a 10% polyacrylamide gel containing 8 M urea followed by PhosphorImager analysis.