Template and primer requirements for DNA Pol θ-mediated end joining

DNA Pol theta-mediated end joining (TMEJ) is a microhomology-based pathway for repairing double-strand breaks in eukaryotes. TMEJ is also a pathway for nonspecific integration of foreign DNAs into host genomes. DNA Pol theta shares structural homology with the high-fidelity replicases, and its polymerase domain Pol theta has been shown to extend ssDNA without an apparent template. Using oligonucleotides with distinct sequences, we find that with Mg2+ and physiological salt concentrations, human Pol theta has no terminal transferase activity and requires a minimum of 2 bp and optimally 4 bp between a template/primer pair for DNA synthesis. Pol theta can tolerate a mismatched base pair at the primer end but loses >90% activity when the mismatch is 2 bp upstream from the active site. Pol theta is severely inhibited when the template strand has a 3' overhang within 3-4 bp from the active site. In line with its TMEJ function, Pol theta has limited strand-displacement activity, and the efficiency and extent of primer extension are similar with or without a downstream duplex.