Nucleic Acids Research, 2012, Vol. 40, No. 14 6781 Figure 5. Binding afﬁnity from the upstream DNA as well as a short 50 ﬂap is necessary for Dna2 displacement by FEN1. Dna2 (25 nM) was pre-incubated with the experimental substrate prior to the addition of increasing concentrations of FEN1 (6.25, 12.5 or 25 nM as indicated in the ﬁgure headings). (A) shows Dna2 and FEN1 binding competition for the 30 nt double-ﬂap substrate (U2:T3:D2.30), the 50-tail substrate (T4:D2.30) and the 5 nt gap-ﬂap (U3:T3:D2.30). (B) shows Dna2 and FEN1 binding competition for the 15 nt double-ﬂap substrate (U2:T3:D2.15) and the 50-tail (T4:D2.15). (C) shows Dna2 and FEN1 binding competition for the 5 nt gap-ﬂap (U3:T3:D2.15) and 2 nt gap-ﬂap (U4:T3:D2.15) having a 15 nt 50 ﬂap. (D) shows the graphical quantitation of the relative percent Dna2 bound to the substrate in the absence of FEN1 or with increasing concen- trations of FEN1 as deﬁned in the ‘Materials and Methods’ section. In (A)–(C) the position of the Dna2–substrate complex and FEN1–substrate complex are indicated to the left and/or right of the ﬁgure. In (A), the FEN1–Dna2–substrate complex is indicated to the left of the ﬁgure. ‘++’ represents the maximum concentration of FEN1 used. In order to determine whether the 1 nt upstream 30 ﬂap was necessary to generate the co-binding complex mode, we pre-bound hDna2 to 5 nt gap-ﬂap substrate, with a 30 nt 50 ﬂap, followed by the addition of increasing con- centrations of competing FEN1 (Figure 5A). hDna2 alone bound to the gap-ﬂap substrate is shown in lane 11 and hFEN1 alone in lane 14. Similar to the 30 nt 50-tail com- petition, when titrated into the reaction, hFEN1 was unable to displace hDna2 from the gap substrate (lanes 12 and 13). Similar to the 30 nt 50-tail structure, the addition of hFEN1 slightly increased the percent DNA substrate bound by hDna2 (graphically represented in Figure 5D, ‘30 nt 50 Flap 5 nt Gap’). Displacement of human Dna2 by FEN1 requires upstream DNA Considering that hFEN1 was unable to effectively displace hDna2 from the long 50 ﬂap substrates, we asked whether the hDna2 displacement by hFEN1 on the intermediate double-ﬂap structure resulted from protein–protein inter- actions or the shift in hDna2-binding afﬁnity based on substrate characteristics. In a model for displacement of hDna2 based on one protein disengaging the other, hFEN1 alters the conformation of hDna2, so that it leaves an intermediate ﬂap 50 ﬂap structure. In the binding afﬁnity displacement model, the substrate ﬂap base properties favor hFEN1 leading to hDna2 displace- ment. To test the protein–protein displacement model, we competed pre-bound hDna2 with hFEN1 on the 15 nt 50-tail structure (Figure 5B). hDna2 alone bound to the 15 nt 50-tail substrate is shown in lane 6, hFEN1 alone in lane 9 and hFEN1 titrated into pre-bound hDna2 reac- tions in lanes 7 and 8. While hFEN1 displaced the pre-bound hDna2 from the 15 nt double-ﬂap structure, minimal hDna2 displacement was visualized from the 15 nt 50-tail structure (compare Lane 3 with Lane 8, re- spectively, and see Figure 5D; ‘15 nt 50-Tail’), providing evidence against the protein–protein interaction model of displacement. To test for displacement based on substrate binding afﬁnity, we utilized the 15 nt gap-ﬂap structures that provided an intermediate hFEN1-binding afﬁnity platform relative to the high afﬁnity double ﬂap and low