6778 Nucleic Acids Research, 2012, Vol. 40, No. 14 Figure 2. Dna2 demonstrates reduced binding for intermediate 50-tail structures relative to double ﬂaps. (A) shows Dna2 binding to the 50-tail substrates having a 50 ﬂap of 15 nt (T2:D1.15), 20 nt (T2:D1.20) or 30 nt (T2:D1.30). The double-ﬂap substrates have a 50 ﬂap of 15 nt (U1:T1:D1.15), 20 nt (U1:T1:D1.20) or 30 nt (U1:T1:D1.30). Binding was measured by EMSA using increasing Dna2 concentrations (3.1, 6.25 or 12.5 nM). (B) shows Dna2 cleavage of 50-tail substrates having a 50 ﬂap of 5 nt (T2:D1.5), 10 nt (T2:D1.10), 15 nt (T2:D1.15), 20 nt (T2:D1.20) or 30 nt (T2:D1.30). Cleavage was measured by denaturing gel electrophoresis using increasing concentrations of Dna2 (0.6, 1.25 or 2.5 nM). (C) shows the graphical quantitation of 12.5 nM Dna2 from (A). In (A), the position of the Dna2–substrate complex is indicated to the left of the ﬁgure. ‘++’ represents the maximum concentration of Dna2 used. ‘DF’ represents the lane containing the double-ﬂap structure. In (B), the position of the radiolabeled oligonucleotide is indicated to the left of the ﬁgure. The experimental 50-tail substrate conﬁguration is shown above each ﬁgure where ‘n’ represents the length of the 50 ﬂap. double-ﬂap substrates, compare Figure 3B (lane 3 and 10) to Figure 1A (lane 28), respectively. We next assessed the impact of the upstream single- stranded gap on the cleavage activity of the two nucleases. Prior results have shown that the 30 ﬂap of the upstream primer signiﬁcantly inﬂuences scFEN1 nuclease activity while scDna2 cleavage remains unchanged (4,20,36). hFEN1 cleavage was greatly reduced with the removal of the upstream 30 ﬂap relative to the double-ﬂap structure (Figure 3C, compare lanes 1–4 with lanes 5–8). hDna2 cleavage efﬁciency was similar for 30 nt 50-tail (lanes 9–12), 5 nt gap-ﬂap (lanes 13–16), 2 nt gap-ﬂap (lanes 17–20) and the double-ﬂap (lanes 21–24) structures. On the long ﬂap structures, both hFEN1 and hDna2 cleavage efﬁciencies were consistent with their yeast homologs. The minimal binding characteristics changed for hDna2 on substrates with short ﬂaps (Figure 2A). Therefore, we examined the impact of short ﬂaps on hDna2 cleavage. hDna2 cleavage of the 15 nt 50-tail structure (Figure 3D, lanes 1–4) was signiﬁcantly less efﬁcient than the cleavage of the 5 nt gap-ﬂap (lanes 5–8), 2 nt gap-ﬂap (lanes 9–12) and double-ﬂap structures (lanes 13–16). Both the binding and cleavage results suggest that hDna2 uses upstream DNA to stabilize binding and improve cleavage on short ﬂap structures. Quantitation of the relative percent of 50-tail, 5 nt gap-ﬂap, 2 nt gap-ﬂap and double-ﬂap DNA substrates cleaved by 0.63 nM hDna2 normalized to the 30 nt double-ﬂap structure is shown in Figure 3E labeled ‘50-Tail’, ‘5 nt Gap’, ‘2 nt Gap’ and ‘Double Flap’, respectively.