6776 Nucleic Acids Research, 2012, Vol. 40, No. 14 Table 1. Oligonucleotide sequences Oligo Length (nt) Sequence Upstream (50–30) U1 26 U2 26 U3 20 U4 23 Downstream (50–30) D1.2 25 D1.5 28 D1.10 33 D1.15 38 D1.20 43 D1.30 53 D2.15 41 D2.30 56 Template (30–50) T1 49 T2 51 T3 26 CGCCAGGGTTTTCCCAGTCACGACCA CGACCGTGCCAGCCTAAATTTCAATT CGACCGTGCCAGCCTAAATT CGACCGTGCCAGCCTAAATTTCA GTCGTTTTACAACGACGTGACTGGG GCCGTCGTTTTACAACGACGTGACTGGG CACTGGCCGTCGTTTTACAACGACGTGACTGGG TAATTCACTGGCCGTCGTTTTACAACGACGTGACTGGG TTAGTTAATTCACTGGCCGTCGTTTTACAACGACGTGACTGGG TTCACGCCTGTTAGTTAATTCACTGGCCGTCGTTTTACAACGACGTGACTGGG ATACGCGCTATAACCCCCGTCCACCCGACGCCACCTCCTGC TTCTACTTCCAATTGATACGCGCTATAACCCCCGTCCACCCGACGCCACCTCCTGC GCGGTCCCAAAAGGGTCAGTGCTGGGCAAAATGTTGCTGCACTGACCCG GCTGGCACGGTCGGATTTAAAGTTAGGGCAGGTGGGCTGCGGTGGAGGACG GGGCAGGTGGGCTGCGGTGGAGGACG a 1 nt 30 overhang and varied lengths of 50 ﬂaps, called a double-ﬂap substrate. Using an EMSA, we compared the binding of hDna2 to substrates with increasing 50 ﬂap lengths (Figure 1A). These experiments were done in the absence of Mg2+ and ATP to prevent nuclease and helicase activities, respectively. hDna2 showed negligible binding to 2 and 5 nt 50 ﬂap substrates (lanes 1–8) but improved binding afﬁnity as the ﬂap length increased from 10 to 30 nt (lanes 9–24). In addition to the predom- inant hDna2 band, additional super-shifted bands, presumed to represent multiple proteins bound likely re- sulting from protein–protein interactions, were visualized on all of the double-ﬂap substrates in a concentration-de- pendent fashion. Consistent with our prior results (15), hFEN1 bound substrates containing 50 ﬂaps from 5 to 30 nt with high afﬁnity (lanes 26–30) but exhibited a slight decrease in binding for the 2 nt ﬂap (lane 25). Quantitation of the percent DNA substrate bound by 12.5 nM hDna2 or 6.3 nM hFEN1 is shown in Figure 1C. In contrast with the scDna2 cleavage speciﬁcity, it was shown that scFEN1 cleaves Okazaki fragments of varying 50 ﬂap lengths with similar efﬁciency (4). We now asked whether the human FEN1 and Dna2 would cleave double- ﬂap substrates with similar characteristics as the yeast homologs. We incubated hFEN1 and hDna2 with the varying 50 ﬂap Okazaki fragment substrates in the presence of Mg2+ and ATP. Consistent with scDna2, hDna2 nuclease activity was not detected on the 2 nt (data not shown) or 5 nt ﬂaps (Figure 1B, lanes 1–4). hDna2 cleavage progressively increased as the 50 ﬂap was lengthened from 10 to 20 nt (lanes 5–8, 9–12 and 13–16, respectively). hDna2 cleaved the 20 and 30 nt ﬂaps with similar efﬁciency (lanes 13–16 and 17–20, re- spectively). Consistent with the previous reports about scFEN1, hFEN1 cleaved substrates with 50 ﬂaps of 2, 5, 10, 15, 20 and 30 nt similarly (lanes 21–25). Quantitation of the percent DNA substrate cleaved by 0.63 nM hDna2 or 0.13 nM hFEN1 is shown in Figure 1D. Dna2 demonstrates reduced binding for short 50 tail structures relative to double ﬂaps Since hDna2 showed reduced binding afﬁnity to sub- strates containing intermediate length 50 ﬂaps (15 nt), we assessed the minimal 50 ﬂap length requirement for stable hDna2 binding. We measured hDna2 binding to sub- strates with various 50 ﬂap lengths in the absence of the upstream double-stranded (ds) DNA, referred to here as a 50-tail structure (Figure 2A). Signiﬁcantly, hDna2 demonstrated lower binding afﬁnity for the 15 nt 50-tail structure relative to the 15 nt double-ﬂap structure (compare lanes 4 and 5). This suggests a role for the upstream DNA region in Dna2 binding stability. Conversely, hDna2 bound the 20 nt 50-tail structure with similar afﬁnity to the 20 nt double ﬂap (compare lanes 9 and 10) and bound the 30 nt 50-tail with slightly higher afﬁnity than the 30 nt double-ﬂap substrate (compare lanes 14 and 15). Quantitation of the percent DNA 50- tail substrate versus the double-ﬂap substrates bound by 12.5 nM hDna2 is shown in Figure 2C. At the maximum concentration used, stable hDna2 binding was visualized neither for the 5 nor 10 nt 50-tail structures (data not shown). hFEN1 binding was not visualized for any of the 50-tail structures (Figure 5A, lane 9 and Figure 5B lane 9, and data not shown). We then asked whether this relative decrease in binding afﬁnity for the short 50-tail substrate would translate to a comparable decrease in nuclease activity. We measured the hDna2 nuclease activity on the 50-tail substrate with ﬂaps ranging from 5 to 30 nt (Figure 2B). hDna2 cleavage was not visualized on the 5 nt 50-tail substrate (lanes 1–4), while cleavage efﬁciency improved with increasing ﬂap length for the 10, 15, 20 and 30 nt 50-tail structures (lanes 5–8, 9–12, 13–16 and 17–20, respectively). Notably, while the cleavage efﬁciency trend was similar, hDna2 cleavage was reduced on short 50-tail structures relative to short double-ﬂap structures.