Structural basis of long-range to short-range synaptic transition in NHEJ

Abstract

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.

Structural insights into the role of DNA-PK as a master regulator in NHEJ

“…The coiled-coil domain of XRCC4 interacts with tandem BRCT domains in the C-terminal region of DNA ligase IV (black). This forms a long-range synaptic complex as reported in Chen et al (2021a). E Autophosphorylation of DNA-PKcs at ABCDE and possibly other sites, likely in trans leads to a conformational change that causes release of the DSB ends by DNA-PKcs.…”

“…Also shown is the position of the conserved forehead domain (892-1289, bright green) and the NUC194 domain (residues 1815-2202, dark green), the YRPD motif (residues 2772-2784, dark blue), the FRB domain (residues 3582-3675, bright blue) and the positions of the PQR, ABCDE, S3205 and T3950 phosphorylation sites (red triangles). B Two views of DNA-PKcs (from PDB 7LT3) (Chen et al, 2021a), rotated by 90°, colored as in A. The YRPD motif is shown in dark blue, most clearly visible in the side view, indicated by an arrow.…”

To Join or Not to Join: Decision Points Along the Pathway to Double-Strand Break Repair vs. Chromosome End Protection

“…Ku binding is traditionally associated with repair by NHEJ whereas MRN is associated with HR-mediated repair. While still not completely understood, recent work, in both yeast and mammals, suggest that the recruitment and binding of these sensors is context dependent and not mutually exclusive (Langerak et al, 2011;Ingram et al, 2019;Chen et al, 2021). Instead, it is the subsequent steps that determine the displacement of these factors to promote HR, NHEJ or end protection.…”

“…To initiate NHEJ, the Ku70/80 heterodimer (hereafter referred to as Ku) and the DNA-PK catalytic subunit (DNA-PKcs) are recruited to damage sites to generate the DNA-PK holoenzyme (Gell and Jackson, 1999;Singleton et al, 1999;Jette and Lees-Miller, 2015). DNA-PK bridges the DNA ends creating a long-range synapse (Graham et al, 2016;Chen et al, 2021). Additional proteins X-Ray Repair Cross Complementing 4 (XRCC4), XRCC4-like factor (XLF) and DNA ligase 4 (LIG4) are recruited to align and ligate the DNA ends (Blackford and Jackson, 2017).…”

A New Assay Capturing Chromosome Fusions Shows a Protection Trade-off at Telomeres and NHEJ Vulnerability to Low Density Ionising Radiation

“…Chromosome stability requires that DNA double-strand breaks (DSBs) are repaired by canonical NHEJ or homologous recombination. NHEJ is an efficient pathway directly resealing DSB ends, most often accurately (1)(2)(3)(4). In line with other DNA repair pathways, NHEJ repair sometimes comes with errors.…”