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. 2010 Aug 25;285(45):34922–34931. doi: 10.1074/jbc.M110.165902

FIGURE 4.

FIGURE 4.

5′ Flap threading reduces dissociation of FEN1 in the presence of a competitor substrate. FEN1 dissociation was measured by EMSA as described under the “Experimental Procedures.” Reactions were initiated by incubating FEN1 with the experimental substrate for 10 min. Ten minutes after reaction initiation, increasing concentrations of competitor substrate were added. A, shows the FEN1 remaining bound to a 5′ 27 nt flap experimental substrates (U2:T2:D2.27) 10 min after the addition of the competitor substrates (2.5×, 5×, 10×, 25×, and 50× molar excess relative to experimental substrate) having (a) a 27 nt flap (U2:T2:D2.27) (lanes 3–7), (b) an upstream 1 nt 3′ flap with no downstream 5′ flap (U2:T2:U2.0) (lanes 10–14), and (c) a 30 nt ssDNA segment (D2.F) (lanes 17–21). Lanes 1, 8, and 15 show the substrate alone controls. Lanes 2, 9, and 16 show experimental substrate bound by FEN1 in the absence of competitor substrate. B shows the FEN1 remaining bound to a 53 nt 5′ biotinylated flap experimental substrate (U1:T1:D1.53B) 10 min after the addition of increasing concentrations of a 53 nt flap competitor substrate (U1:T1:D1.53) (7.5×, 15×, 30×, 75×, and 150× molar excess relative to experimental substrate) where (a) no streptavidin was added to the reaction (lanes 3–7), (b) streptavidin was added 10 min after reaction initiation (lanes 10–14), or (c) streptavidin was added 10 min before reaction initiation (lanes 17–21). Lane 1 shows the experimental substrate alone control. Lanes 8 and 15 show the streptavidin-bound experimental substrate controls. Lanes 2, 9, and 16 show the experimental substrates bound by FEN1 in the absence of competitor substrate. Quantitation of the percent experimental substrate bound by FEN1 and standard deviation based on at least three independent EMSA results are shown below the figure. The position of the experimental substrate alone and FEN1, experimental substrate complexes are indicated to the left of the figure. The competitor substrate configurations are shown above the figure. In B, a “B” in the oligonucleotide sequence indicates the location of the 5′ biotin, and the black-circled “B” represents streptavidin-bound biotin.