Figure 4.
EhPCNA enhances the nick‐sealing activity of EhDNAligI and ΔPIP and restores the activity of ΔDBD. Reaction containing 500 fmol of [γ‐32P]‐labeled DNA substrate, 50 fmol of EhDNAligI and deletion mutants, and 150 fmol of EhPCNA or HsPCNA was incubated at room temperature for 0, 1, 2, 4, 8, and 16 min, aliquots were quenched at the indicated times adding stop buffer and loaded into a 15% polyacrylamide/8M urea gels. (A) Enzymatic activity of full‐length; lanes 1–6, analysis of time course activity only with full‐length; lanes 7–12, enzymatic activity when EhPCNA has been added; lanes 13–18, enzymatic activity with HsPCNA. (B and C) Time course activity with ∆PIP and ∆DBD deletion mutant as explained in A. Data are presented as pmol of ligated product; these values are plotted for EhDNAligI and deletion mutant as a function of incubation time. A diagram showing the double‐stranded nicked substrate is present in the upper part of the figure. The radioactive label at 5′ end of the 21 mer is indicated by an asterisk.