Figure 7. AOA2 mutants Sen1 RNA binding mechanism.

(A) Structural overlay of an Alphafold model of human SETX helicase domain (blue) with the CtSen1 RNA complex (tan).

(B) Alphafold model of the hSETX helicase domain showing the location of mapped AOA2 and ALS4 mutations

(C) Two AOA2 mutations map to the RNA binding cleft. Human equivalent positions are shown below the CtSen1 numbering in parentheses and the CtSen1 structure is displayed.

(D) Fluorescence polarization ssRNA binding. Binding to ssRNA was conducted using enzyme titration and monitoring fluorescence polarization from Sub8 (Supplementary Table 3). Error bars reflect SD from 3 replicates.

(E) ATPase activity. Proteins (5 nM) were incubated with Sub6 (Supplementary Table 3) and ATP (1 mM) at 5 μM for 15 min at 37 °C. The L1551W mutant had no measurable activity. Error bars are SD from 3 replicates, **p<0.01, ****p<0.0001.

(F) RNA—DNA unwinding activity. Proteins (1 nM) were incubated with 50 nM Sub7 (Supplementary Table 3), ATP (1 mM) and MgCl₂ (2 mM). FAM signal at 10 min was used to compare the unwinding activities of Sen1^Hel mutants. The L1551W mutant had no measurable activity. Error bars are SD from 3 replicates, **p<0.01, ****p<0.0001.

(G) Purified Sen1^Hel proteins used in panels B–D.

(H) Model for Sen1 autoinhibition and RNA translocation.