Cryo-EM visualization of DNA-PKcs structural intermediates in NHEJ

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.

Unravelling the complexities of DNA-PK activation by structure-based mutagenesis.

“…The DNA binding cradle interactions are required for both types of kinase activation. The reduction in 2056 phosphorylation with both the HHH and DEB mutants suggest that these DNA binding motifs are required for full kinase activation; moreover, the severe celllular phenotypes associated with these mutations are consistent with impaired formation of the DNA end protection complex which in cryo-EM structures is associated with full kinase activation 10 , 15 , 16 . Mutation of the DEB revealed that it is the failure of the DEB interaction with the DNA end that results in the partial and transient activation of DNA-PK; studies of DNA-PK activation by hairpin ends predicted this mechanism 10 .…”

“…The enormous 4128 residue DNA-PKcs polypeptide is composed largely of well-ordered HEAT repeats with the relatively small lipid-like kinase domain positioned at the C-terminus of the polypeptide. Previously, a psi-pred analysis of DNA-PKcs predicted that ~200 residues that includes the functionally critical "ABCDE" autophosphorylation sites 23 was likely disordered; this is the same region that includes the DEB helix that can be visualized in both monomeric and dimeric structures [9][10][11][12][15][16][17][18]25 . Exactly what promotes the initial transition from a disordered region to the lone alpha helix DEB is not clear.…”

“…It is important to note that the DEB helix is disordered when ABCDE sites (2609–2647) are phosphorylated. Phosphorylated ABCDE sites are repositioned by binding basic residues in the M-HEAT region, disrupting the closely connected DEB helix (2737–2765) and opening of the DNA binding cradle to promote end-processing by Artemis 10 , 16 .…”

“…What is still unclear is how DSBs are shuttled between the two dimer forms (for DSBs that require both nucleolytic and fill in end processing) and how the XLF-dependent dimers progress to ligation competent short-range complex. Although elegant studies from He and colleagues have observed complexes that arise from ABCDE phosphorylation within the XLF-dependent dimer 16 , it is unclear whether this intermediate complex progresses to the ligation competent short-range NHEJ complex. Thus, there remains a significant gap in knowledge regarding how NHEJ progresses through these many structurally distinct complexes to promote NHEJ’s iterative manner of repair in living cells.…”

“…Although it has been known for decades that the DNA dependent protein kinase is fully activated when DNA-PKcs associates with a DNA double-stranded end bound to Ku [6][7][8] , the molecular events that promote activation have not been defined. Recently, there has been an explosion of new structural and functional information (from cryo-EM and mutational studies) of NHEJ complexes that provide snapshots of distinct steps of the joining mechanism [9][10][11][12][13][14][15][16][17][18] . These studies have highlighted several DNA-protein interactions that could potentially impact kinase activation (Fig.…”