Nucleic Acids Research, 2012, Vol. 40, No. 14 6777 Figure 1. Dna2 binding correlates to cleavage on Okazaki fragment double-ﬂap structures. Dna2 and FEN1 binding and nuclease activity of double-ﬂap substrates having a 50 ﬂap of 2 nt (U1:T1:D1.2), 5 nt (U1:T1:D1.5), 10 nt (U1:T1:D1.10), 15 nt (U1:T1:D1.15), 20 nt (U1:T1:D1.20) or 30 nt (U1:T1:D1.30) were measured by EMSA and denaturing gel electrophoresis, respectively, as described in the ‘Material and Methods’ section. (A) shows Dna2 (3.1, 6.25 and 12.5 nM) and FEN1 (6.25 nM) binding to the double-ﬂap substrates. (B) shows Dna2 (0.6, 1.25 and 2.5 nM) and FEN1 (0.13 nM) cleavage of the double-ﬂap substrates. (C) shows the graphical quantitation of 12.5 nM Dna2 and 6.3 nM FEN1 from (A) and (D) shows the quantitation of 0.63 nM Dna2 and 0.13 nM FEN1 from (B). In (A), the position of the Dna2–substrate complex and FEN1– substrate complex are indicated to the left of the ﬁgure. In (B), the position of the radiolabeled oligonucleotide is indicated to the left of the ﬁgure. The experimental substrate conﬁguration is shown above each ﬁgure where ‘n’ represents the length of the 50 ﬂap. On short ﬂap substrates, an upstream region improves Dna2 binding while stable FEN1 binding requires the full double-ﬂap structure In addition to its nuclease function, Dna2 also possesses 50–30 helicase activity, although the reported helicase function of hDna2 is much weaker compared to scDna2 (33,34). Nevertheless, Dna2 may have evolved to recog- nize substrates with a gap between the upstream synthesis primer and downstream 50 ﬂap. In order to assess the inﬂuence of the 30 end of the upstream primer on Dna2 binding afﬁnity, we measured hDna2 binding to substrates with gap-ﬂap structures, i.e. having an upstream primer separated by a gap from the ﬂap base (Figure 3A). We were surprised to ﬁnd that compared to the 30 nt double- ﬂap structure (lanes 19–21), hDna2 demonstrated slightly higher binding afﬁnity for the gap-ﬂap structures con- taining either a 5 nt (lanes 5–7) or 2 nt single-stranded gap (lanes 12–14). Conversely, hFEN1 exhibited a sub- stantial loss in binding afﬁnity as the upstream primer single-stranded gap was increased from 2 nt (lanes 9–11) to 5 nt (lanes 2–4) relative to the double-ﬂap structure (lanes 16–18). The amount of stably bound hFEN1 was decreased as the single-stranded gap size was increased, as evident from the smearing between the hFEN1–DNA complex band and the free substrate band indicative of FEN1 dissociation during electrophoresis. To further explore the combined impact of ﬂap length and upstream DNA structure on binding afﬁnities of hFEN1 and hDna2, we measured afﬁnity for 15 nt 50 gap-ﬂap structures (Figure 3B) containing a 5 nt (lanes 5–7) or 2 nt (lanes 12–14) single-stranded gap. Consistent with results using the double-ﬂap and 50-tail structures, hDna2-binding afﬁnity for the 15 nt ﬂap was reduced 2-fold with respect to the 30 nt gap-ﬂap structures. hFEN1 binding afﬁnity was generally low for the 30 and 15 nt gap-ﬂap structures with 5 and 2 nt gaps. Figure 3A showing the 30 nt gap-ﬂap structures with 5 nt (lanes 2–4) and 2 nt (lanes 9–11) gaps can be compared with Figure 3B showing the 15 nt gap-ﬂap structures with 5 nt (lanes 2–4) and 2 nt (lanes 9–11) gaps. Irrespective of whether the ﬂap length was 15 or 30 nt, binding afﬁnity to the double-ﬂap conﬁguration was much higher. For the 15 nt gap and