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

Significance Using oligonucleotides comprised solely of purines or pyrimidines, we find that human Polθ is a bona fide template-dependent DNA polymerase and has no terminal transferase activity under physiological conditions. Polθ requires a minimum of 2 bp and optimally 4 bp between a template/primer pair for efficient and processive DNA synthesis. Polθ polymerase activity is inhibited by a 3′-overhang on the template strand. Consistent with in vivo observations of DNA deletion during TMEJ, 3′-exonucleases or 3′-flap endonucleases are likely recruited to remove such hindrances when microhomology is internal to the 3′ ends. In contrast to a previously report, we observed limited strand displacement activity by Polθ. Because Pol θ lacks a proofreading exonuclease activity and is error prone, minimal strand displacement can be only beneficial for TMEJ.