Structural and functional insights into the interaction between Ku70/80 and Pol X family polymerases in NHEJ

Nat Commun. 2025 May 6;16(1):4208. doi: 10.1038/s41467-025-59133-2.

Abstract

Non-homologous end joining (NHEJ) is the main repair pathway for double-strand DNA breaks (DSBs) in mammals. DNA polymerases lambda (Pol λ) and mu (Pol μ), members of the Pol X family, play a key role in this process. However, their interaction within the NHEJ complexes is unclear. Here, we present cryo-EM structures of Pol λ in complex with the DNA-PK long-range synaptic complex, and Pol μ bound to Ku70/80-DNA. These structures identify interaction sites between Ku70/80 and Pol X BRCT domains. Using mutants at the proteins interface in functional assays including cell transfection with an original gap-filling reporter, we define the role of the BRCT domain in the recruitment and activity of the two Pol X members in NHEJ and in their contribution to cell survival following DSBs. Finally, we propose a unified model for the interaction of all Pol X members with Ku70/80.

MeSH terms

  • Cryoelectron Microscopy
  • DNA / metabolism
  • DNA Breaks, Double-Stranded
  • DNA End-Joining Repair*
  • DNA Polymerase beta* / chemistry
  • DNA Polymerase beta* / genetics
  • DNA Polymerase beta* / metabolism
  • DNA Polymerase beta* / ultrastructure
  • DNA-Binding Proteins / metabolism
  • DNA-Directed DNA Polymerase* / chemistry
  • DNA-Directed DNA Polymerase* / genetics
  • DNA-Directed DNA Polymerase* / metabolism
  • DNA-Directed DNA Polymerase* / ultrastructure
  • HEK293 Cells
  • Humans
  • Ku Autoantigen* / chemistry
  • Ku Autoantigen* / genetics
  • Ku Autoantigen* / metabolism
  • Ku Autoantigen* / ultrastructure
  • Models, Molecular
  • Protein Binding

Substances

  • Ku Autoantigen
  • DNA-Directed DNA Polymerase
  • DNA Polymerase beta
  • DNA polymerase X
  • DNA polymerase mu
  • DNA polymerase beta2
  • Xrcc6 protein, human
  • DNA
  • DNA-Binding Proteins