Figure 6. The SAM-key interaction with the ATPase domain is essential for nucleosome remodeling by Fun30.
(A, B, C, D) A mutant (Fun30 I367R, C374R [Fun30-ICRR]) with a defect in the SAM-key protrusion I interface binds normally to DNA and nucleosome, and behaves therefore similar to Fun30∆SAM. (A) DNA binding of Fun30-ICRR and quantification in gel shifts. DNA binding to a 247-bp dsDNA construct. Left: gel shift in native gel with Fun30 WT and Fun30-ICRR and DNA stained with ethidium bromide. Representative gel of n = 4 biological replicates. Right: Coomassie staining shows equal amounts of WT and mutant protein were used in both DNA- (A) and nucleosome-binding experiments (C) (quantified from band intensity). (B) Quantification of DNA binding as in Fig 2A. n = 4 replicates, filled circles indicate mean, error-bars depict SD. Individual datapoints of replicates are shown in Fig S6E. (C, D) Nucleosome binding by Fun30-ICRR and quantification. (C) Binding to 100W0 nucleosomes of Fun30 WT and Fun30-ICRR. Gel shift shown by fluorescence imaging of the labeled histone H2A (46-C-D550) in native gel. Representative gel of n = 4 biological replicates. (D) Quantification of nucleosome binding as in Fig 2B. n = 4 replicates, filled circles indicate mean, error-bars depict SD. Individual datapoints of replicates are shown in Fig S6F. (E, F, G) Fun30 nucleosome remodeling requires the SAM-key interaction with protrusion I of the ATPase catalytic domain. (E) Nucleosome-sliding assay (with 100W0 end-positioned nucleosomes and labelled H4 as in Fig 3B) shows the nucleosome-sliding defect of Fun30-ICRR. Representative gel of n = 2 biological replicates and n = 4 technical replicates. (F) Eviction assay (labelled H4 as in Fig 3F) shows nucleosome eviction defect of Fun30-ICRR. Representative gel of n = 4 biological replicates. (G) DNA-stimulated ATPase assay as in Fig 3I but with Fun30-ICRR. Unlike Fun30 WT that reaches a kcat of 1.5 s−1, Fun30 ICRR only reaches a kcat of 0.2 s−1 comparable with Fun30∆SAM. n = 4 replicates shown by filled circles, central line indicates mean, error-bars depict SD.
Source data are available for this figure.
