FIGURE 6.

ATP binding and hydrolysis-coupled conformational changes. A and B, models of the Mot1 ATPase in the open and closed forms, respectively. The open complex model was obtained using the open structure of Rad54 from S. solfataricus (Protein Data Bank code 1Z6A) (28), and the closed form was obtained using the D. rerio Rad54 structure (Protein Data Bank code 1Z3I) (26). Experimentally identified catalytic residues from each RecA motif are colored in cyan (domain 1A) and red (domain 2A). In the open form, the red catalytic residue is hidden behind the green helices (see supplemental Movies 1–3). In the closed form, the cyan and red residues are brought together to establish a catalytically active ATP binding pocket. Images were generated using PyMOL software (The PyMOL Molecular Graphics System, Version 1.5.0.4, Schrödinger, LLC). C and D, model of the Mot1 ATPase in the same orientation as A and B. The ATPase was docked onto DNA by aligning the N-terminal RecA domain of each model with the N-terminal RecA domain of SsoRad54 in the SsoRad54-DNA complex (1Z6A). The nucleotide-dependent cleavage in domain 2B observed using the 6Fe probe is shown in cyan. The extent of shading corresponds to the mapped cleavage site ±S.D. obtained from independent experiments. E, model of the TBP-DNA co-complex showing the locations of DNA gaps that affect Mot1 catalytic activity. Color coding of DNA base pairs is the same as in Fig. 2E in which pink represents the locations of DNA gaps that confer reduced TBP-DNA dissociation activity, red is a gap with reduced Mot1 and TBP dissociation from DNA, purple is similar to red but the effect of the gap falls just short of statistical significance using p < 0.05 as the cutoff (see text), and the loss of the green bases generates a template that supports enhanced TBP-DNA dissociation compared with the WT.