Structural Basis for Pol theta-Helicase DNA Binding and Microhomology-Mediated End-Joining
DNA double-strand breaks (DSBs) present a critical threat to genomic integrity, often precipitating genomic instability and oncogenesis. Repair of DSBs predominantly occurs through homologous recombination (HR) and non-homologous end joining (NHEJ). In HR-deficient cells, DNA polymerase theta (Pol{theta}) becomes critical for DSB repair via microhomology-mediated end joining (MMEJ), also termed theta-mediated end joining (TMEJ). Thus, Pol{theta} is synthetically lethal with BRCA1/2 and other HR factors, underscoring its potential as a therapeutic target in HR-deficient cancers. However, the molecular mechanisms governing Pol{theta}-mediated MMEJ remain poorly understood. Here we present a series of cryo-electron microscopy structures of the Pol{theta} helicase domain (Pol{theta}-hel) in complex with DNA containing 3'-overhang. The structures reveal the sequential conformations adopted by Pol{theta}-hel during the critical phases of DNA binding, microhomology searching, and microhomology annealing. The stepwise conformational changes within the Pol{theta}-hel subdomains and its functional dimeric state are pivotal for aligning the 3'-overhangs, facilitating the microhomology search and subsequent annealing necessary for DSB repair via MMEJ. Our findings illustrate the essential molecular switches within Pol{theta}-hel that orchestrate the MMEJ process in DSB repair, laying the groundwork for the development of targeted therapies against the Pol{theta}-hel.