9588 Nucleic Acids Research, 2012, Vol. 40, No. 19 Figure 1. Puriﬁcation and ATPase activity of wild-type and mutant Fml1 Cs. (A) Schematic of Fml1 showing the position of its helicase motifs, point of truncation in Fml1 C and residues mutated to disrupt its ATPase activity. (B) Coomassie blue-stained SDS polyacrylamide gel showing puriﬁed wild-type and mutant His-tagged Fml1 C proteins. (C) Comparison of the amount ATP hydrolysed by wild-type and mutant Fml1 C proteins. See ‘Materials and Methods’ section for further details. Figure 2. Binding and unwinding X-12 by wild-type and mutant Fml1 Cs. (A) Electrophoretic mobility shift assay showing the binding of X-12 by increasing amounts of Fml1 C (lanes b–e: 0.05, 0.5, 5 and 10 nM), Fml1 CK99R (lanes g–j: 0.05, 0.5, 5 and 10 nM) and Fml1 CD196N (lanes l–o: 0.05, 0.5, 5 and 10 nM). See ‘Materials and Methods’ section for further details. (B) Comparison of X-12 unwinding by Fml1 C (lanes b–d: 0.05, 0.5 and 5 nM) Fml1 CK99R (lane e: 5 nM) and Fml1 CD196N (lane f: 5 nM). The schematics represent X-12 and its dissociation products with the asterisk indicating the position of the 50-end 32P-label. See ‘Materials and Methods’ section for further details. producing a single complex at low protein concentrations and two additional more retarded complexes at higher concentrations (Figure 2A). As shown previously, wild-type Fml1 C readily dissociates X-12 into ﬂayed duplexes in the presence of Mg2+ and ATP (Figure 2B, lanes b–d). In contrast, both mutant proteins are unable to dissociate X-12 (Figure 2B, lanes e and f). These data show that K99R and D196N mutations have no affect on Fml1 C’s ability to bind junction DNA but do abolish its ability to unwind them. Fml1"CK99R and Fml1"CD196N are defective for fork regression In addition to unwinding junctions, Fml1 C can also catalyse the regression of model replication forks (15). This process involves branch point migration through homologous DNA and could potentially be promoted by protein–DNA interactions in the absence of ATP hy- drolysis. Indeed, previous studies of ATPase-deﬁcient mutants of human FANCM have suggested that this