6780 Nucleic Acids Research, 2012, Vol. 40, No. 14 on 50 ﬂap substrates (Figure 4A). To measure hDna2 dis- placement by hFEN1, we pre-bound hDna2 to a 30 nt double-ﬂap structure prior to the addition of increasing concentrations of competing hFEN1, as described in the ‘Materials and Methods’ section. hDna2 alone bound to the substrate appeared as the predominant hDna2 bound band (Labeled ‘Dna2’) and lesser super-shifted multimeric bands (lane 2). At the lowest concentration of hFEN1 used, the super-shifted hDna2 bands were reduced in in- tensity, implying multimeric hDna2 displacement from the substrate while the predominant hDna2–DNA complex was not dissociated (lane 3). In addition, a new band appeared corresponding to a hFEN1–DNA complex (Labeled ‘FEN1’). As the hFEN1 concentration was increased (lanes 4–6), a large fraction of the hDna2– DNA alone complex remained visible and a new band appeared retarded more than the predominant hDna2– DNA band but less than the super-shifted hDna2 bands. This new super-shifted band (Labeled ‘FEN1 and Dna2’) was visualized in neither the substrate alone (lane 1), hDna2 alone (lane 2), nor hFEN1 alone titration reactions (lanes 7–10) suggesting that hFEN1 and hDna2 can bind concurrently. Quantitation of the percent DNA 30 nt double-ﬂap substrate bound by hDna2 in the presence of competing hFEN1 normalized to the percent DNA sub- strate bound by hDna2 alone is shown in Figure 5D indicated as ‘30 nt Double Flap’. The inability of hFEN1 to displace hDna2 was surprising because our previous results with nucleases from yeast showed that scFEN1 efﬁciently removes scDna2 from double-ﬂap structures (17,25). Because we observed a signiﬁcant difference between the yeast and human systems, we considered that the variance might have resulted from differences in experimental conditions. In our previous yeast binding and competition assays, Ca2+ was used in place of Mg2+ to inhibit nuclease activity. In our human protein binding and competition assays, we excluded divalent metal ions and added a small amount of EDTA to inhibit nuclease activity. To distin- guish between differences in experimental conditions, we repeated our prior yeast competition experiments using the human competition assay conditions. Consistent with prior results, scFEN1 displaced scDna2 in a concen- tration-dependent fashion in the presence of EDTA and independent of the Ca2+ (Supplementary Figure S1). Initial results showed that hDna2-binding afﬁnity is highly sensitive to 50 ﬂap length while hFEN1 binding is relatively insensitive (Figure 1A). We considered that the human system might have adopted a ﬂap length threshold below which hFEN1 could displace pre-bound hDna2. To test this, we repeated the competition assay as in Figure 4A with a 15 nt 50 ﬂap (Figure 4B). Similar to the 30 nt ﬂap reaction conditions, we allowed hDna2 to pre-bind the 15 nt double-ﬂap substrate prior to the addition of hFEN1. hDna2 alone binding is shown in lane 2. hFEN1 was then titrated into the reaction in com- petition with hDna2 (lanes 3–6) or alone (lanes 7–10). The relative hDna2 displacement from the 15 nt double ﬂap is shown graphically in Figure 5D as ‘15 nt Double Flap’. In contrast to the 30 nt ﬂap results, hFEN1 demonstrated the ability to displace hDna2 from the 15 nt 50 ﬂap in a Figure 4. FEN1 preferentially displaces Dna2 from intermediate length double-ﬂap structures. Dna2 (25 nM) was pre-incubated with the ex- perimental substrate prior to the addition of increasing concentrations of FEN1 (3.1, 6.25, 12.5 or 25 nM in lanes 3–6). Lane 1 shows the substrate alone, lane 2 shows Dna2 bound without FEN1 and lanes 7– 10 show FEN1 alone bound to the substrate at the same concentrations as in 3–6. (A) shows Dna2 and FEN1 binding competition for the 30 nt double-ﬂap substrate (U2:T3:D2.30). (B) shows Dna2 and FEN1 binding competition for the 15 nt double-ﬂap substrate (U2:T3:D2.15). 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 (A), the FEN1–Dna2–substrate complex is indicated to the left of the ﬁgure and to the right of lane 6 with a dashed arrow. In (B), the quantitation of the percent substrate bound by hDna2 or hFEN1 is shown below the ﬁgure. concentration-dependent fashion. Quantitation of the percent substrate bound by hDna2 or hFEN1 is shown below the ﬁgure. In addition, the super-shifted hFEN1– hDna2 co-binding complex visualized in the 30 nt 50 double ﬂap EMSA no longer appeared in the 15 nt 50 double ﬂap competition experiment. Dna2 and FEN1 co-binding requires upstream DNA Prior reports show that optimal FEN1 substrate binding requires an upstream 1 nt 30 ﬂap (37). To further charac- terize substrate properties that enable hFEN1 and hDna2 substrate co-binding, we competed hDna2, pre-bound to a 30 nt 50-tail substrate, with hFEN1 (Figure 5A). When titrated into the reaction (lanes 7 and 8), hFEN1 was unable to compete hDna2 away from the 30 nt 50-tail. The hDna2/hFEN1 co-binding complex was not visualized on the 50-tail substrate (compare lane 8 to lane 3). The addition of hFEN1 to the reaction enabled a slight increase in the relative amount of DNA substrate bound by hDna2 alone (graphically represented in Figure 5D; ‘30 nt 50-Tail’).