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. 2017 Jun 16;8:15847. doi: 10.1038/ncomms15847

Figure 4. Stimulation of the ZRANB3 nuclease activity by PCNA.

Figure 4

(a) Stimulation of FEN1 and ZRANB3 nuclease activities by PCNA. FEN1 or ZRANB3 nucleases were incubated with the DNA substrate and increasing concentrations of PCNA (0.375, 0.75, 1.5 and 3 μM), in the presence (ZRANB3) or absence (FEN1) of ATP. (b) Domain structure of ZRANB3. Conservation of the PIP box and the APIM motif residues among ZRANB3 proteins are shown in the alignments below. Mutated conserved residues in the PIP box and the APIM motif are indicated by arrows. (c) Interactions of the ZRANB3 PIP box and APIM motif with PCNA. Biotinylated PIP box and APIM motif peptides were bound to streptavidin beads and incubated with recombinant PCNA. The interaction was assayed by Western blotting with PCNA antibody. Mutations of the conserved residues (Q519A, F525A, and F526A in PIP* and F1075A in APIM*) abrogated the interaction with PCNA. (d) Effect of PCNA (0.375, 0.75, 1.5 and 3 μM) on ZRANB3 nuclease activity assayed with the wild-type ZRANB3 and ZRANB3 PCNA binding mutants (ZRANB3-PIP*, ZRANB3-ΔAPIM and ZRANB3-PIP*ΔAPIM). (e) Quantification of nuclease activities in the presence of increasing concentrations of PCNA. Shown are averages of five reactions. s.d.’s are shown as error bars.