Human DNA polymerase θ harbors a DNA end-trimming activity critical for DNA repair

Cancers with hereditary defects in homologous recombination rely on DNA polymerase θ (pol θ) for repair of DNA double-strand breaks. During end-joining, pol θ aligns microhomology tracts internal to 5′-resected broken ends, after which an unidentified nuclease trims the 3′-end before synthesis can occur. We report here that such a nuclease activity, which differs from the proofreading activity often associated with DNA polymerases, is intrinsic to the polymerase domain of pol θ. Like the DNA synthesis activity, the nuclease activity requires conserved metal-binding residues, metal ions and dNTPs, and is inhibited by ddNTPs or chain-terminated DNA. Our data indicate that pol θ repurposes metal ions in the polymerase active site for endonucleolytic cleavage, and that the polymerase-active and end-trimming conformations of the enzyme are distinct. We reveal a nimble strategy of substrate processing that allows pol θ to trim or extend DNA depending on the DNA repair context. DNA polymerase θ is essential for the double strand break repair pathway termed theta-mediated end-joining. Microhomology pairing of two DNA strands generates 3′-unpaired ends. The nuclease responsible for cleaving 3′-ends has been elusive. Zahn et al . show that such an activity resides within Pol θ.