Fig. 4: CHD6 possesses direct DNA binding activity.
For all statistical analysis in this figure, 2-way ANOVAs followed by Tukey post hoc tests were carried out. ns = not significant (p > 0.05), *p < 0.05. ** p < 0.01. ***p < 0.001.****p < 0.0001. Source data are provided as a Source Data file. Panel a. Coomassie stain of indicated MBP-tagged purified proteins. Panel b. Increasing amounts of hCHD6 aa 1090-1619 wildtype, ΔDBD1 or ΔDBD2 point mutants were incubated with double-stranded DNA and resolved by electrophoretic mobility shift assay (EMSA). Please note that we attribute the absence of a discrete DNA complex band in the highest concentrations of ΔDBD1 (despite loss of free probe signal) to the overall weak but still measurable affinity of ΔDBD1 for DNA (compared to WT and ΔDBD2) that results in a less stable complex with a higher off-rate causing free probe signal to smear across the middle section of the gel at higher concentrations, rather than form a discrete band. Panel c Workflow for fluorescence polarization-based DNA binding assay. Panel d MBP-fused proteins from (a) were titrated into fluorescently labelled double-stranded DNA and resulting change in depolarization was measured; fluorescence polarization line graphs show geometric means ± CI95%; n = 3. Dissociation constants (KD) were determined by fitting the data to a logistic function and identifying the point on the model with the largest change in fluorescence polarization. Panel e Wildtype and indicated point mutants of hCHD6 aa 1090-1619 (from a) were assayed as in (d); graph shows geometric means ± CI95%; n = 3.
