Nucleic Acids Research, 2012, Vol. 40, No. 14 6779 Figure 3. Dna2 binding is improved by the upstream region for short ﬂap structures while stable FEN1 binding requires the full upstream region. (A) shows Dna2 or FEN1 binding to the 5 nt gap-ﬂap (U3:T3:D2.30), 2 nt gap-ﬂap (U4:T3:D2.30) and double-ﬂap (U2:T3:D2.30) substrates with 30 nt 50 ﬂaps. (B) shows Dna2 or FEN1 binding to the 5 nt gap-ﬂap (U3:T3:D2.15) and 2 nt gap-ﬂap (U4:T3:D2.15) structures with 15 nt 50 ﬂaps. Binding was measured in (A) and (B) by EMSA using increasing Dna2 and FEN1 concentrations (3.1, 6.25 or 12.5 nM). (C) shows FEN1 cleavage of the 5 nt gap-ﬂap (U3:T3:D2.30) and double-ﬂap (U2:T3:D2.30) structures (lanes 1–8) and Dna2 cleavage of the 50-tail (T4:D2.30), 5 nt gap-ﬂap (U3:T3:D2.30), 2 nt gap-ﬂap (U4:T3:D2.30) and double-ﬂap (U2:T3:D2.30) structures (lanes 9–24) with 30 nt 50 ﬂaps. (D) shows Dna2 cleavage of the 50-tail (T4:D2.15), 5 nt gap-ﬂap (U3:T3:D2.15), 2 nt gap-ﬂap (U4:T3:D2.15) and double-ﬂap (U2:T3:D2.15) structures with 15 nt 50 ﬂaps. Nuclease activity was measured by denaturing gel electrophoresis. (E) shows the graphical quantitation of the relative cleavage of 0.63 nM Dna2 from (C) and (D) as deﬁned in the ‘Materials and Methods’. In (A) and (B) the position of the substrate alone, Dna2–substrate complex and FEN1–substrate complex are indicated to the left of the ﬁgure. In (C) and (D) the position of the radiolabeled oligonucleotide is indicated to the left of the ﬁgure. The experimental substrate conﬁgurations are shown above ﬁgures (A)–(D). Displacement of human Dna2 by human FEN1 requires a short 50 ﬂap It was previously reported that, after cleaving, scDna2 remains unproductively bound to the base of the ﬂap, requiring displacement by scFEN1 (17,25). Though both FEN1 and Dna2 preferentially bind to the ﬂap base, their modes of binding appear to need different attributes of the 50 and 30 ﬂaps, as well as the DNA surrounding the ﬂap base junction. Based on substrate characteristics preferred by the two nucleases, we used DNA-binding competition assays to determine how these proteins acted sequentially