Cryo-EM of NHEJ supercomplexes provides insights into DNA repair

Abstract

Non-homologous end joining (NHEJ) is one of two critical mechanisms utilized in humans to repair DNA double-strand breaks (DSBs). Unrepaired or incorrect repair of DSBs can lead to apoptosis or cancer. NHEJ involves several proteins, including the Ku70/80 heterodimer, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), X-ray cross-complementing protein 4 (XRCC4), XRCC4-like factor (XLF), and ligase IV. These core proteins bind DSBs and ligate the damaged DNA ends. However, details of the structural assembly of these proteins remain unclear. Here, we present cryo-EM structures of NHEJ supercomplexes that are composed of these core proteins and DNA, revealing the detailed structural architecture of this assembly. We describe monomeric and dimeric forms of this supercomplex and also propose the existence of alternate dimeric forms of long-range synaptic complexes. Finally, we show that mutational disruption of several structural features within these NHEJ complexes negatively affects DNA repair.

Keywords: DNA repair; DNA-PK; NHEJ; cryo-EM; long-range synaptic complexes; non-homologous end joining.

Figure 1.. The structure of the assembly of DNA-PK, XRCC4, XLF, and the BRCT tandem repeats of LigIV

(A) The overall structure of DNA-PK, XRCC4, XLF, and LigIV BRCT tandem repeats in a monomeric form. DNA-PKcs is shown in gray, Ku70 in orange, Ku80 in green, XLF in pink, XRCC4 in blue, DNA in yellow, and LigIV BRCT repeats in red. The cryo-EM map to 4.3-Å resolution is shown as a gray transparent surface. The inset shows an enlarged view of the interaction between the BRCT1 domain of LigIV (red) and Ku70/80 (orange and green, respectively). (B) The overall structure of DNA-PK, XRCC4, XLF, and LigIV BRCT tandem repeat monomer rotated by 180° and proteins colored according to (A). The inset shows an enlarged view of the interaction between the stalk of XLF (pink) with Ku70 (orange).

Figure 2.. The structure of the dimeric NHEJ supercomplex containing DNA-PK, XRCC4, XLF, and the BRCT tandem repeats of LigIV

(A) The overall assembly of the supercomplex XLF-mediated DNA-PK dimer in two orthogonal orientations. DNA-PKcs is shown in gray, Ku70 in orange, Ku80 in green, XLF in pink, XRCC4 in blue, DNA in yellow, and LigIV BRCT repeats in red. The cryo-EM map to 4.1-Å resolution is shown as a gray transparent surface. (B) Two orientations of the dimer interface with loop 1 (L1; residues 898–901) shown in pink, loop 2 (L2; residues 946–950) in yellow, loop 3 (L3; residues 2567–2572) in blue, and loop 4 (L4; residues 2578–2583) in purple, with protomer A in light gray and protomer B in darker gray. (C) A top view of the DNA-PKcs components of the dimer in two shades of gray. The kinase domains (yellow) are shown in close proximity to the PQR (red) and ABCDE (blue) phosphorylation clusters on the opposite protomer (red).

Figure 3.. Long-range synaptic complexes

(A) NHEJ supercomplex dimer (XLF-mediated DNA-PK dimer [PDB: 7NFC], this work). DNA-PKcs is shown in gray, Ku70 in orange, Ku80 in green, XLF in pink, XRCC4 in blue, DNA in yellow, and LigIV BRCT repeats in red. The DNA is shown below the structure in yellow, with the distance between the DNA ends indicated. (B) The Ku80-mediated DNA-PK dimer (Chaplin et al., 2021) with DNA-PKcs is shown in gray, Ku70 in orange, Ku80 in green, and the DNA in yellow. The DNA is shown in yellow below the structure, with the distance between the DNA ends indicated.

Figure 4.. Disruption of DNA-PK dimer interfaces prevents recombination

(A) The structure of the NHEJ dimer supercomplex with DNA-PKcs is shown in gray, Ku70 in orange, Ku80 in green, XLF in pink, XRCC4 in blue, DNA in yellow, and LigIV BRCT repeats in red. Inset: enlarged view of the supercomplex dimer interface with L1 (residues 898–900) is shown in cyan and L3 (residues 2567–2570) in blue. (B) The structure of the previous DNA-PK dimer (PDB: 6ZHE) with DNA-PKcs is shown in gray, Ku70 in orange, and Ku80 in green (Chaplin et al., 2021). Inset: enlarged view of the dimer interface, with the four basic residues interacting with the C terminus of Ku80 shown in red. (C) Episomal end-joining assays analyzing the effects of DNA-PKcs dimer interface mutations. DNA-PKcs-deficient V3 cells were co-transfected with the 290-RFP/CFP (red and cyan fluorescent protein) coding joint (left) or 289-RFP/CFP signal joint (right) substrates with no R (RAG1+RAG2), R, or R plus wild-type or mutant DNA-PKcs constructs as indicated. The wild type is shown in white, 898–900 > A (L1) in cyan, 2569–2571 > A (L3) in blue, 898–900 > A and 4XKR > A in purple, and 4XKR > A in red. Cells were analyzed by flow cytometry; the percentage of cells expressing CFP/RFP is indicated as percent recombination. Results were compiled from at least four experiments; ****p < 0.0001, ***p = 0.0002. (D) Immunoblot analyses of whole-cell extracts of DNA-PKcs-deficient 293T cells transiently transfected with no DNA-PKcs, wild-type DNA-PKcs, or mutant DNA-PKcs as indicated. 48 h after transfection, cells were treated with 40 nM calicheamicin and 1 mM okadaic acid for 30 min or left untreated. DNA-PKcs phospho-specific antibodies utilized include anti-phospho-S2056 (working concentration, 1:1000; Abcam 18192) and a rabbit anti-phospho-T2609 reagent, a generous gift from Dale Ramsden (working concentration, 1:500) (Neal et al., 2016).

Figure 5.. Comparison between DNA-PKcs in the monomeric and dimeric NHEJ supercomplexes

(A) Two orientations comparing the structure of DNA-PKcs in the monomer with DNA-PKcs in the dimer. DNA-PKcs in the dimer is colored according to the sequence schematic below the structures, with the N-terminal arm in purple, circular cradle in red-orange, FAT (FRAP [FKBP12-rapamycin-associated protein]) domain in teal, and kinase domain in yellow. DNA-PKcs in the monomer is shown in gray for only the head domain because of the rest of the structure being similar to the dimeric DNA-PKcs structure. The two new helices present in the supercomplex dimeric structure are colored in green and labeled on the sequence schematic. The loops forming the dimer interface in Figure 2 are also labeled and colored on the sequence schematic. Inset: rotation of the head domain by 90° to show the twisting of the head domain in the dimer structure compared with the monomer. (B) A close-up view of the ordered helix (residues 2737–2765) in green and the DNA in blue. (C) Episomal end-joining assays were performed as described in Figure 3. (D) Enlarged view comparing the interaction shown in the dimer (left) between the N-terminal arm shown in purple and the FAT domain in teal with the lack of an interaction shown in the monomer (right). Map density is shown as gray mesh.

Figure 6.. Overall model of NHEJ

DNA-PKcs is shown in gray, Ku70 in orange, Ku80 in green, XLF in pink, XRCC4 in blue, DNA in yellow, and LigIV in red. PDB and references are given below the structures.