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TitleCryo-EM visualization of DNA-PKcs structural intermediates in NHEJ.
Journal, issue, pagesSci Adv, Vol. 9, Issue 22, Page eadg2838, Year 2023
Publish dateJun 2, 2023
AuthorsSiyu Chen / Alex Vogt / Linda Lee / Tasmin Naila / Ryan McKeown / Alan E Tomkinson / Susan P Lees-Miller / Yuan He /
PubMed AbstractDNA 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 ). ...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 ). 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 ). 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.
External linksSci Adv / PubMed:37256947 / PubMed Central
MethodsEM (single particle)
Resolution3.6 - 14.0 Å
Structure data

28731

8ez9

EMDB-28731, PDB-8ez9:
Dimeric complex of DNA-PKcs
Method: EM (single particle) / Resolution: 5.67 Å

28732

8eza

EMDB-28732, PDB-8eza:
NHEJ Long-range complex with PAXX
Method: EM (single particle) / Resolution: 4.39 Å

28733

8ezb

EMDB-28733, PDB-8ezb:
NHEJ Long-range complex with ATP
Method: EM (single particle) / Resolution: 8.9 Å

EMDB-28734: DNA-PKcs monomer in dimer DNA-PKcs complex
Method: EM (single particle) / Resolution: 4.3 Å

EMDB-28735: DNAPKcs-Ku-LigIV(BRCT)-PAXX-DNA subcomplex in PAXX-LR complex
Method: EM (single particle) / Resolution: 3.6 Å

EMDB-28736: XRCC4-XLF-XRCC4 subcomplex of PAXX-LR complex
Method: EM (single particle) / Resolution: 14.0 Å

EMDB-28737: DNA-PKcs without N-terminus in PAXX-LR-ATP complex
Method: EM (single particle) / Resolution: 5.0 Å

EMDB-28738: DNAPKcs-Ku-DNA subcomplex in PAXX-LR-ATP complex
Method: EM (single particle) / Resolution: 5.9 Å

EMDB-28739: XRCC4-XLF-XRCC4 subcomplex in LR-ATP complex
Method: EM (single particle) / Resolution: 12.0 Å

Chemicals

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

ChemComp-MG:
Unknown entry

Source
  • homo sapiens (human)
KeywordsDNA BINDING PROTEIN / Dimer / Kinase / Complex / NHEJ / DNA BINDING PROTEIN/DNA / DNA BINDING PROTEIN-DNA complex

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