was constructed from two nearly identical plasmids pG46 and pG68 as described (32,33). In brief, differently sized complementary single-strand gaps were introduced into each plasmid using the nicking endonucleases Nt.BbvCI and Nb.BbvCI. One of the plasmids (pG46) was then radiolabelled with 32P at its free 50 and the other (pG68) linearized by XhoI digestion. The two plasmids were then annealed and model replication forks separated from non-annealed DNA by agarose gel electrophoresis followed by puriﬁcation using a QIAquick Gel Extraction Kit (Qiagen). DNA-binding assays Reaction mixtures (20 ml) contained 0.5 nM labelled X-12 in buffer (25 mM Tris–HCl, pH 8.0, 1 mM DTT, 100 mg/ml BSA, 6% glycerol) plus wild-type or mutant Fml1 C protein as indicated. Reactions were incubated on ice for 15 min and then loaded onto a 4% native polyacrylamide gel in low ionic strength buffer (6.7 mM Tris–HCl, pH 8.0, 3.3 mM sodium acetate, 2 mM EDTA) that had been pre-cooled at 4 C. Gels were run for 1 h and 45 min at 160 V with continuous buffer recirculation. Gels were then dried on 3 mm Whatman paper and analysed by Phosphor Imaging using a Fuji FLA3000 and Image Gauge software. DNA unwinding assay Reaction mixtures (20 ml) contained 0.5 nM labelled sub- strate DNA in buffer (50 mM Tris–HCl, pH 8.0, 1 mM DTT, 100 mg/ml BSA, 6% glycerol) plus 2.5 mM MgCl2, 5 mM ATP and protein as indicated. Reactions were incubated at 30 C for 30 min and then terminated by adding one-ﬁfth volume of stop mix (2.5% so- dium dodecyl sulphate, 200 mM EDTA, 10 mg/ml protein- ase K) and incubating for a further 15 min at 37 C to deproteinize the mixture. Reaction products were separated by electrophoresis through a 10% native poly- acrylamide gel at 200 V in TBE buffer. The gel was dried on 3 mm Whatman paper and analysed by Phosphor Imaging using a Fuji FLA3000 and Image Gauge software. Fork regression assay Reaction mixtures (40 ml) contained 5 nM of the plasmid-sized labelled fork substrate in buffer (50 mM Tris–HCl, pH 8.0, 1 mM DTT, 100 mg/ml BSA, 6% glycerol) plus 2.5 mM MgCl2, 5 mM ATP and 5 nM of wild-type or mutant Fml1 C. Reactions were incubated for 30 min at 30 C after which samples were taken and digested with the indicated restriction enzymes (note that the excess magnesium ions in the restriction enzyme buffers effectively stops the fork regression reaction). The digested samples were separated by electrophoresis through a 10% native polyacrylamide gel. The gel was dried on 3 mm Whatman paper and analysed by Phosphor Imaging using a Fuji FLA3000 and Image Gauge software. Nucleic Acids Research, 2012, Vol. 40, No. 19 9587 RESULTS DNA-binding and ATPase activities of Fml1 Full-length Fml1 is insoluble when produced in E. coli, and therefore to study its biochemical activities in vitro, we puriﬁed a C-terminally truncated form of it (Fml1 C), which contains the entire helicase domain (Figure 1A and B). Fml1 C is able to fully complement the hyper- sensitivity of a fml1 mutant to the alkylating agent MMS when over-expressed, demonstrating that it retains its core biological activity (15). Fml1 C binds well to a range of different branched DNA structures in vitro, including model replication forks, D-loops and HJs (15). It also binds well to ssDNA but poorly to linear dsDNA (Supplementary Figure S1A). This suggests that Fml1 C’s ability to bind branched DNA could simply be a consequence of its ssDNA-binding activity. However, Fml1 C binds well to the static HJ X0, which is composed of duplex DNA that is not prone to extensive base-pair ‘breathing’ (34), indicating a genuine speciﬁcity for branched DNA molecules over linear duplex DNA (Supplementary Figure S1A). Indeed, its binding to ssDNA could be due to interaction with DNA secondary structures rather than to ssDNA itself. Consistent with its DNA-binding prefer- ence, Fml1 C exhibits a DNA-dependent ATPase activity that is stimulated more strongly by ssDNA than by dsDNA (Supplementary Figure S1B). ATPase-deﬁcient mutants of Fml1 To investigate the importance of Fml1’s ATPase activity, we changed conserved residues in motif I (K99R) and II (D196N) of its helicase domain (Figure 1A and Supplementary Figure S2). C-terminally truncated forms of each mutant protein were puriﬁed by the same protocol used previously for wild-type protein (Figure 1A and B) (15). The puriﬁed mutant proteins were then compared to wild-type for their ability to hydrolyse ATP in the presence of ssDNA (Figure 1C). During the 30-min reaction, wild-type Fml1 C hydrolysed 2900 pmol of ATP. In contrast, both mutant proteins hydrolysed only 80– 90 pmol, which was only just above background levels. The same inability to hydrolyse ATP was also observed when dsDNA was used instead of ssDNA (data not shown). These data show that K99R and D196N mutations in Fml1 C disable its DNA-dependent ATPase activity. Fml1"CK99R and Fml1"CD196N exhibit normal junction DNA binding but are deﬁcient in dissociating synthetic HJs To establish the in vitro capabilities of the Fml1 CK99R and Fml1 CD196N proteins they were tested for their ability to bind and dissociate a synthetic HJ (X-12). X-12 is made by annealing four 50-mer oligonucleotides and contains a 12-bp homologous core, in which the junction point is free to migrate and 18–20 bp heterol- ogous arms that prevent the junction from spontaneously dissociating. To assess binding to X-12, an eletrophoretic mobility shift assay (EMSA) was used. Both wild-type and mutant Fml1 C proteins bind to X-12 equally well