Fig. 2: FeS cluster loss induces a conformational change that affects DNA binding.
From: The iron–sulphur cluster in human DNA2 is required for all biochemical activities of DNA2
a Representative EMSA of DNA2/DNA complexes using a 5′ flap DNA substrate (FAM-labelled) at 4 nM final concentration and increasing amounts of DNA2 wild-type and C396S, and quantification of DNA binding expressed in % (n = 3 independent experiments; error bars, SD). b EMSAs as in a, using 20 and 100 nM of DNA2 wild-type and FeS cluster-deficient variants (n = 3 independent experiments; error bars, SD). c Relative distance between free DNA and DNA2/DNA complexes in EMSAs shown in b, where distance for wild-type DNA2 samples is set to 100% (n = 3 independent experiments; error bars, SD). Statistical analysis: ordinary one-way ANOVA (****p < 0.0001). d Purified DNA2 wild-type, C136S and C396S were incubated with Proteinase K for 1 min. The reactions were stopped and visualised by SDS–PAGE and InstantBlue staining (top). The band intensities were analysed by a line scan (bottom). The position of the proteolytic product unique to digested wild-type DNA2 is indicated by the arrow. The asterisk represents a heat shock protein contaminant, identified by mass spectrometry. The full gel can be found in Supplementary Fig. 2d.
