DNA double-strand breaks (DSBs) are the most cytotoxic form of DNA damage, with their aberrant repair linked with carcinogenesis 1,2 . The conserved error-prone Non-Homologous End-Joining (NHEJ) pathway plays a key role in determining the effects of DSB-inducing agents used to treat cancer as well as the generation of antibody and T cell receptor diversity 2,3 . Here, we applied single-particle cryo-electron microscopy (EM) to visualize two key DNA-protein complexes formed by NHEJ factors. Ku, DNA-PKcs, LigIV-XRCC4, and XLF form a Long-range synaptic complex, in which the DNA ends are held ~115 Å apart. Two DNA end-bound Ku-DNA-PKcs subcomplexes are linked by DNA-PKcs-DNA-PKcs interactions and a LigIV-XRCC4-XLF-XRCC4-LigIV scaffold. The relative orientation of the DNA-PKcs molecules suggests a mechanism for auto-phosphorylation in trans, leading to dissociation of DNA-PKcs and transition into the Short-range synaptic complex. Within this complex, the Ku-bound DNA ends are aligned for processing and ligation by the XLF-anchored scaffold, and a single LigIV catalytic domain is stably associated with a nick between the two Ku molecules, suggesting that joining of both strands of a DSB involves both LigIV molecules.

No abstract

DNA double-strand breaks (DSBs), one of the most cytotoxic forms of DNA damage, can be repaired by the tightly regulated nonhomologous end joining (NHEJ) machinery (Stinson and Loparo and Zhao et al. ). Core NHEJ factors form an initial long-range (LR) synaptic complex that transitions into a DNA-PKcs (DNA-dependent protein kinase, catalytic subunit)–free, short-range state to align the DSB ends (Chen et al. ). Using single-particle cryo–electron microscopy, we have visualized three additional key NHEJ complexes representing different transition states, with DNA-PKcs adopting distinct dimeric conformations within each of them. Upon DNA-PKcs autophosphorylation, the LR complex undergoes a substantial conformational change, with both Ku and DNA-PKcs rotating outward to promote DNA break exposure and DNA-PKcs dissociation. We also captured a dimeric state of catalytically inactive DNA-PKcs, which resembles structures of other PIKK (Phosphatidylinositol 3-kinase-related kinase) family kinases, revealing a model of the full regulatory cycle of DNA-PKcs during NHEJ.