(A) Diagram of AP lyase assay. (B) NEIL2 outcompetes APE1 on AP sites generated from 5’-[32P]-U-containing ssDNA (51 nt) by UDG. Amounts of NEIL2-His6 or APE1 are just sufficient to completely cleave the AP site (lane 7 of Figure 4—figure supplement 3A,B). The NEIL2 product retains a 3’P and migrates faster than 3’OH-terminated APE1 product. When both NEIL2 and APE1 are present, only the NEIL2 product is generated (lane 5). S, substrate; P, product. (C) Concentration gradient and product accumulation curves of APE1 and NEIL2 on 5’-[32P]-U-containing dsDNA (35 nt) in the presence of UDG. The volumes of NEIL2 (68 ng/µl) and APE1 (0.005 U/µl) used are listed in the figure. (D) NEIL2 competes with APE1 on 5’-[32P]-U-containing dsDNA (35 nt) in the presence of UDG. The reactions contained either 0.005 U (Low APE1) or 0.02 U APE1 (High APE1) and increasing amounts of NEIL2-His6. Lanes 2 and 3 contain respectively 4 μl NEIL2 (68 ng/μl) and 0.02 U APE1. The APE1 cleavage pattern was converted to the NEIL2 pattern with increasing amounts of NEIL2. S, substrate; P, product. (E) Incorporation of [α-32P]-dCTP by Polβ for products generated by APE1, NEIL2, and NEIL2 and PNKP on di-deoxynucleotide (ddC)-modified oligonucleotide in the presence of UDG. P, product. (F) Incorporation of [α-32P]-dCTP by Polβ in the presence of UDG and APE1 as a function of NEIL2 (68 ng/μl, 0, 1, 2, 5, 10 μl) and constant PNKP (127 ng/μl, 2 μl). As the NEIL2 product increasingly dominates the reaction, Polβ incorporation decreases. The number under each band gives the intensity relative to that in the first lane. P, product.