Cryo-EM structure of DNA polymerase θ helicase domain in complex with inhibitor novobiocin

DNA double-strand breaks (DSBs) are highly toxic lesions that occur during the cellular metabolic process. DNA Polymerase theta (Polθ) is an error-prone polymerase that has been implicated in the repair of chromosome breaks, recovery of broken replication forks, and translesion synthesis. The inhibition of Polθ activity has been implicated in killing HR-deficient tumor cells in vitro and in vivo . We present the first biochemical evidence that the antibiotics novobiocin (NVB) noncompetitively inhibit ATP hydrolysis by the ATPase domain of the Polθ helicase domain (Polθ-HLD). We report the Cryo-EM structure of apo dimeric Polθ helicase domain (Polθ-HLD), and the first inhibitor occupied Polθ-HLD structure. Our structure identifies a non-canonical novobiocin binding pocket, distinct from the canonical site that partially overlaps with the ATP in the ATPase domain. Comparison with the homolog helicase Hel308-DNA duplex complex suggests that the novobiocin competitively binds to a triangle hub on the DNA translocation pathway and blocks the ssDNA binding and translocation. Furthermore, the first dimeric structure of Polθ-HLD also provides a structural framework for revealing the microhomology-mediated end-joining mechanism. Our results demonstrate that the inhibitor-occupied structure combined with rational, structure-based drug design will undoubtedly accelerate the discovery of potent inhibitors with better efficacy and target selectivity to human Polθ.