Polθ steric-gate variants exhibit robust RNA synthesis activity. (A) Superposition of Polθ:DNA/RNA and Polθ:DNA/DNA crystal structures. Superposition of Polθ:DNA/RNA (red; PDB ID: 6XBU) and Polθ:DNA/DNA (pink; PDB ID: 4x0q) with incoming ddGTP reveals significant conformational changes in the thumb and fingers subdomain of Polθ when bound to A-form DNA/RNA. (B) Denaturing gel showing efficient extension of RNA/DNA by WT Polθ in the presence of dNTPs. (C) Denaturing gel showing premature termination by WT Polθ on RNA/DNA in the presence of NTPs. (D–F) Denaturing gels showing efficient DNA-dependent RNA synthesis by PolθRP1 and PolθRP1 variants on the indicated primer–templates in the presence of NTPs, MgCl2, and indicated NaCl titration. (G) Denaturing gel showing RNase H degradation of the RNA portion of the RNA/DNA hybrid synthesized by PolθRP2. (H) Denaturing gel showing T7 RNAP promoter-dependent synthesis of RNA with an expected length of 95 nt.
