Fig. 5: Mechanistic models of inhibition by AB25583.

a DNA-bound model of Polθ-hel dimer. The bound-DNA with 3′-overhang ssDNA (light and dark gray tubes) was modeled from a homolog structure of HEL308 (PDB ID: 2P6R) by superimposition HEL308 to the Polθ-hel:AB25583 structure (see Supplementary Fig. 6a). AB25583 is shown in spheres with carbon atoms in cyan. Duplex DNA is outside the helicase ring channel, and the 3′-overhang ssDNA passes through the central-channel. b Location of AB25583 in a DNA-bound model. AB25583 lies in the path of the 3′-overhang ssDNA, which is predicted to block DNA translocation. c Comparison of AB25583-bound cryo-EM structure (pink, this study) and the inhibitor-free crystal structure of Polθ-hel (blue, PDB ID: 5AGA). The two structures were superimposed based on the D4 subdomain that mediates the dimerization of Polθ-hel. The largest displacement was observed for the D1 subdomain with up to 9 Å shift in the main-chain atoms between the two structures. d The same comparison of the two structures around the AB25583-binding site as in panel-c, but with a zoom-in view around the rachet helix. In the AB25583-bound structure (pink), the R-helix containing two arginines (R193 and R200) in the D1 subdomain shifted toward the ratchet helix in the D4 subdomain, resulting in tighter packing of D1 and D4 subdomains than that in the inhibitor-free structure (blue).