Skip to main content
. 2018 Oct 5;7:e37774. doi: 10.7554/eLife.37774

Figure 2. Analysis of CtMot1 mutants.

(A) View at the interface between RecA2A (green cartoon), HR1/2 (yellow surface), and lobe 1 (orange/blue surface). Residues analyzed in this study are shown as sticks and labelled accordingly. (B) Cartoon model showing the positions of mutations in the Mot1NTD (red spheres on yellow surface) and in lobe 2 (green spheres on green surface). Left: orientation as in the CtMot1 crystal structure. Right: CtMot1 with ATPase modeled as in the S. cerevisiae Chd1:nucleosome complex, that is the ATP hydrolysis-competent conformation (Farnung et al., 2017). (C) ATPase activity of the mutants. Error bars represent standard deviations from three technical replicates. CtMot1WT is labelled as WT, CtMot1ΔC as ΔC. (D) Electrophoretic mobility shift assay showing ATP-dependent dissociation of Mot1:TBP:DNA and TBP:DNA complexes. All CtMot1 constructs form ternary complexes with labelled DNA and TBP (M:T:D). In the presence of ATP and unlabeled competitor DNA (DNA*), wild-type CtMot1 (WT), L1658/Y1659, R4D, and D1720R mutants fully disrupt M:T:D and T:D complexes (lanes 5, 9, 11, and 13, respectively), whereas CtMot1ΔC (ΔC) is less efficient (lane 7).

Figure 2—source data 1. Raw data from the ATPase activity assay used for Figure 2C and Figure 2—figure supplement 1A.
elife-37774-fig2-data1.xlsx (143.9KB, xlsx)
DOI: 10.7554/eLife.37774.007
Figure 2—source data 2. Raw data from quantification of electrophoretic mobility shift assay used for Figure 2—figure supplement 1B.
elife-37774-fig2-data2.xlsx (18.4KB, xlsx)
DOI: 10.7554/eLife.37774.008

Figure 2.

Figure 2—figure supplement 1. Analysis of CtMot1 mutants.

Figure 2—figure supplement 1.

(A) ATPase activity of the CtMot1 mutants. This panel represents the same data as shown in Figure 2C but normalized against the basal activity of each CtMot1 variant. (B) Dissociation of Mot1:TBP:DNA and TBP:DNA complexes in an electrophoretic mobility shift assay. The assay was performed analogously to the one shown in Figure 2D, but more TBP was added and ATP incubation was shorter (see Materials and Methods). These experimental conditions were chosen so that the reaction does not reach the endpoint to see the differences in dissociation catalyzed by WT versus L1658A/Y1659A, R4D, or D1720R. (C) Quantitation of electrophoretic mobility shift assays as shown in panel B. The bars represent the ratio between the intensity of free DNA and complex bands (Mot1:TBP:DNA and TBP:DNA) in each lane. The reaction without proteins (D) was normalized to 1. T:D - TBP:DNA, WT:T:D-Mot1WT:TBP:DNA, ΔC:T:D–Mot1ΔC:TBP:DNA, L1658A/Y1659A:T:D-Mot1L1658A/Y1659A:TBP:DNA, R4D:T:D-Mot1R4D:TBP:DNA, D1720R:T:D-Mot1D1720R:TBP:DNA. * - reactions performed in the presence of unlabeled competitor DNA. Quantitation was done using ImageJ. Data represent mean values ± standard deviation (n = 6).