interacts with the DNA with speciﬁc polarity showing strong binding to 50 sequences and weak interactions with 30 sequences (32). This polarity guides the orientation of Dna2 so that it has 50 cleavage speciﬁcity. This ability of Dna2 to process the 50 ﬂap but not 30 ﬂap generates a 30-tail structure necessary to initiate recombinational DNA repair and promote telomere maintenance (30). These activities on the pseudo-Y structure imply that Dna2 recognizes dif- ferent features of substrate structure than FEN1 for initial binding, threading and cleavage. Human Dna2 (hDna2) has been reported to have dif- ferent functional characteristics than scDna2, speciﬁcally a much weaker DNA helicase function (33,34). This sug- gested that the sequential interactions of Dna2 and FEN1 differ in the human and yeast systems. In this report, we set out to explore binding and cleavage characteristics of the human nucleases to clarify their interactions during Okazaki fragment processing. MATERIALS AND METHODS Materials Oligonucleotides were synthesized by Integrated DNA Technologies (IDT). DNA substrates were radiolabeled with [a-32P]dCTP (6000 Ci/mmol) purchased from PerkinElmer Life Sciences. The Klenow fragment of DNA polymerase I purchased from Roche Applied Science was used to 30 radiolabel oligonucleotides. All other reagents were purchased from the best commercial sources available. Oligonucleotides Oligonucleotides are listed in Table 1. Upstream primers and downstream ﬂap primers are listed in the 50–30 direc- tion, and templates in the 30–50 direction, for annealing alignment. Oligonucleotides were 30 radiolabeled, an- nealed and puriﬁed for binding and nuclease visualization as previously described (35). Double-ﬂap and gap-ﬂap experimental substrates containing a 30 downstream radiolabeled primer, template and upstream primer were annealed in a ratio of 1:2:4, respectively. Substrates con- taining a 30 radiolabeled downstream 50-tail and template were annealed in a ratio of 1:2. The location of the radiolabel on the experimental substrate downstream ﬂap primer is noted in each ﬁgure with an asterisk. Enzymes Recombinant human FEN1 was cloned into the pET- FCH plasmid, over-expressed in Escherichia coli strain BL21 (DE3)/pLysS and puriﬁed using a C-terminal histi- dine tag as previously described (6). Recombinant human Dna2 was over-expressed using pFastBac HTc vector in baculovirus High Five cells and puriﬁed using a C-terminal FLAG tag as previously described (33). Binding assays FEN1 and Dna2 binding afﬁnities with various experi- mental substrate conﬁgurations were measured using an electrophoretic mobility shift assay (EMSA). All binding Nucleic Acids Research, 2012, Vol. 40, No. 14 6775 assays were performed using a reaction buffer containing 50 mM Tris–HCl (pH 8.0), 2 mM DTT, 0.25 mg/ml bovine serum albumin (BSA), 30 mM NaCl, 20 mM EDTA and 5% glycerol. For non-competition experiments, reactions were initiated by incubating either Dna2 for 10 min or FEN1 for 5 min with the experimental substrate on ice. After the pre-incubation period, the reactions were placed at 37 C for an additional 10 min. For competition reactions, Dna2 was incubated with the experimental sub- strate for 5 min on ice followed by the addition of FEN1 for 5 more min on ice. Reactions were further incubated at 37 C for an additional 10 min. Reactions were then loaded onto a 6% native polyacrylamide gel in 1 TBE and sub- jected to electrophoresis at 200 V for 45 min. Nuclease assays FEN1 and Dna2 nuclease activities were measured by incubating the enzymes with various experimental sub- strates at 37 C for 20 min. Reactions were performed in a reaction buffer containing 50 mM Tris–HCl (pH 8.0), 2 mM DTT, 0.25 mg/ml BSA, 30 mM NaCl, 4 mM MgCl2, 2 mM ATP and 5% glycerol. Reactions were terminated by the addition of 20 ml of 2 termination dye and heating to 95 C for 5 min. Termination dye con- tained 90% formamide (v/v), 10 mM EDTA, as well as xylene cyanole and bromophenol blue markers. Nuclease activity was measured using 15% polyacrylamide, 7 M urea denaturing gels in 1 TBE that were subjected to electrophoresis for 60 min at 80 W. Experimental result analysis For both native and denaturing assays, gel drying and exposure were conducted as previously described (35). Autoradiography images were generated using Storm hardware (GE Healthcare) with Molecular Dynamics PhosphorImager technology and quantiﬁed utilizing Molecular Dynamics ImageQuant software version 5.2. All reactions were performed at least in triplicate and rep- resentative gels are shown. The percent DNA substrate bound by either FEN1 or Dna2 is deﬁned as [bound/ (bound+unbound)] 100. The percent DNA substrate cleaved by either FEN1 or Dna2 is deﬁned as [cleaved/ (cleaved+uncleaved)] 100. Error bars indicate ±stand- ard deviation. The relative DNA substrate cleaved by Dna2 (Figure 3E) is normalized to the amount of 30 nt double-ﬂap DNA substrate cleaved by 0.63 nM Dna2. The relative DNA substrate bound by Dna2 (Figure 5D) is normalized to the amount of DNA substrate bound by 25 nM Dna2 in the absence of FEN1. RESULTS Human Dna2 preferentially binds and processes long ﬂap Okazaki fragment substrates Prior work showed that the cleavage activity of scDna2 on 50 ﬂap substrates improved as the length of the 50 ﬂap increased (23). In order to correlate the binding properties of Dna2 with the cleavage function, we ﬁrst measured the binding characteristics of hDna2 on substrates containing