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 (Pol{theta}) 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 Pol{theta} 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 Pol{theta} helicase domain (Pol{theta}-HLD). We report the Cryo-EM structure of apo dimeric Pol{theta} helicase domain (Pol{theta}-HLD), and the first inhibitor occupied Pol{theta}-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 Pol{theta}-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 Pol{theta}.