Cryo-EM visualization of state transition during non-homologous end-joining (NHEJ).

DNA double-strand breaks (DSBs) are a highly cytotoxic form of DNA damage and the incorrect repair of DSBs is linked to carcinogenesis. The conserved error-prone non-homologous end joining (NHEJ) pathway has a key role in determining the effects of DSB-inducing agents that are used to treat cancer as well as the generation of the diversity in antibodies and T cell receptors. Using single-particle cryo-electron microscopy, we visualized key DNA-protein complexes that are formed by human NHEJ factors. A near-complete NHEJ reaction cycle and a life cycle of DNA-PKcs (catalytic subunit of the DNA-dependent protein kinase) are proposed from the models. The Ku70/80 heterodimer (Ku), DNA-PKcs, XRCC4 and XLF first form a long-range synaptic complex in which the DNA ends are held approximately 115 Å apart. Two DNA end-bound subcomplexes comprising Ku and DNA-PKcs are linked by interactions between the DNA-PKcs subunits and a scaffold comprising LigIV, XRCC4, and XLF. The relative orientation of the DNA-PKcs molecules suggests a mechanism for autophosphorylation in trans, which leads to substantial outward rotation of both Ku and DNA-PKcs to promote DNA double-strand break exposure and DNA-PKcs dissociation. Upon DNA-PKcs dissociation, the long-range synaptic complex transit into the short-range synaptic complex, within which the Ku-bound DNA ends are aligned for processing and ligation by the XLF-anchored scaffold, and a single catalytic domain of LigIV is stably associated with a nick between the two Ku molecules, suggesting that the joining of both strands of a DSB involves both LigIV molecules. In addition, we captured a dimeric state of catalytically inactive DNA-PKcs, which resembles previously determined structures of PIKK family kinases, revealing a model of the full life cycle of DNA-PKcs during NHEJ.