Fig. 1.

Polθ is template-dependent and selects correct dNTP for incorporation in the presence of Mg²⁺. (A) The purity of human DNA Polθ. (B) DNA synthesis on ssDNAs (oligo1–3) of mixed purines and pyrimidines at 37 °C with all four dNTPs. (C) The restricted purine or pyrimidine-only ssDNA cannot be extended by Polθ. (D) The ³²P-labeled pyrimidine strand is extended by Polθ in the presence of the complementary purine strand by 2–3 bp for 10 min. “4” indicates all four dNTPs were present. (E) Both strands of a template/primer pair with terminal 3 bp at the 3′ end can be extended. Polθ incorporates the correct dNTP according to the template sequence. In reactions shown in B–D, the Polθ to DNA molar ratio was 1:10, and in E, it was 1:1.