FIGURE 2.

Threading of the 5′ flap allows for FEN1 cleavage. The comparison of FEN1 binding and cleavage was made using EMSA and denaturing gel electrophoresis as described under “Experimental Procedures.” Reactions were initiated by incubating increasing concentrations of FEN1 (a) (0.16 nm) and (b), (c) (0.16, 0.31, 0.63, 1.25, 2.5 nm) with the 53 nt 5′ biotinylated experimental flap substrate (U1:T1:D1.53B), referred to as the binding reaction. Similar to Fig. 1A, the experimental flap substrate was (a) not streptavidin blocked (lanes 1–2), (b) streptavidin blocked 10 min after the binding reaction initiation (lanes 3–8), or (c) streptavidin blocked 10 min before the binding reaction initiation (lanes 9–14). Lane 1 shows the substrate alone control. Lanes 3 and 9 show the streptavidin-bound substrate controls. Fifteen minutes after the binding reaction initiation, an aliquot from the reaction was extracted and mixed with Mg²⁺, referred to as the cleavage reaction. A, shows FEN1 bound to the experimental substrate in the binding reaction and B, shows the corresponding labeled oligonucleotide and FEN1 cleavage products in the cleavage reaction. In A, the positions of the substrate alone and FEN1-substrate complex are indicated to the left of the figure. A “B” in the oligonucleotide sequence indicates the location of the 5′ biotin, and the black-circled “B” represents streptavidin-bound biotin. In B, the position of the labeled oligonucleotide and FEN1 cleavage products are indicated to the left of the figure. Quantitation of the percent experimental substrate bound (A) and cleaved (B) by FEN1 and standard deviation based on at least three independent EMSA results are shown below each figure.