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. 2016 Dec 27;114(2):304–309. doi: 10.1073/pnas.1610011114

Fig. 4.

Fig. 4.

Zf-GRF DNA binding is critical for APE2 function. (A) 3′-5′ exonuclease assays using a 3′ recessed ssDNA/dsDNA structure with FAM-labeling on the 5′ terminus of the short strand. Zf-GRF mutations of the positive residues in the crescent platform reveal effects on full-length exonuclease activity. (B) Whereas sperm chromatin and hydrogen peroxide were added to mock-depleted egg extracts, WT APE2 or ΔZF APE2 was added to APE2-depleted egg extracts supplemented with sperm chromatin and hydrogen peroxide. After a 45-min incubation, total egg extracts were examined as indicated via immunoblotting analysis. “Endo APE2” represents endogenous APE2. A star indicates a nonspecific protein overlapping with WT Myc-APE2. (C) WT APE2 or ΔZF APE2 was added to APE2-depleted egg extracts supplemented with sperm chromatin and hydrogen peroxide. After a 45-min incubation, chromatin fractions (“chromatin”) were isolated and examined as indicated via immunoblotting analysis. Total egg extracts were analyzed via immunoblotting analysis. (D) WT APE2 or R502E APE2 was added to APE2-depleted egg extracts supplemented with sperm chromatin and hydrogen peroxide. After a 45-min incubation, chromatin fractions and total egg extracts were analyzed via immunoblotting analysis as in C. (E) WT Zf-GRF or R502E Zf-GRF was added to egg extracts, to a final concentration of 35 nM. Egg extracts were then supplemented with sperm chromatin and hydrogen peroxide. After a 45-min incubation, total egg extracts were examined via immunoblotting analysis.