Acknowledgements: JBC thank the I2BC platforms for Interactions of Macromolecules (PIM) and for Crystallization supported by French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INBS-0005. We thank the HTX platform at EMBL, Grenoble. We thank Magali Aumont-Nicaise and Adrian Velazquez-Campoy for their help with ITC data. AG was supported by a CIFRE (ANRT) fellowship. JBC was supported by ANR-20-CE11-0026, ANR-17-CE12-0020 and ANR-21-CE12-0019-01. We also thank the staff from PROXIMA-1, PROXIMA-2a and SWING beamlines for their help in data collection and synchrotron SOLEIL (Saint-Aubin, France) for provision of synchrotron radiation facilities. SJ, MJM, and ER thank Mauro Modesti for providing SNAP tag Ku protein. PC, SB and PF were supported by the Ligue Contre le Cancer as Equipe Labellisée 2018 and ANR-20-CE11-0026. PC is a scientist from INSERM. We acknowledge the TRI imaging facility, member of the national infrastructure France-BioImaging supported by the French National Research Agency (ANR-10-INBS-04). We would like to thank the Imaging Core Facility TRI-IPBS, in particular Antonio Peixoto and Eve Pitot for technical assistance. A.C., A.K. and T.L.B. thank the Wellcome Trust for support of this research through the award of an Investigator Award (200814/Z/16/Z; 2016–2022).Funder: Ligue Contre le Cancer; DOI: https://doi.org/10.13039/501100004099The classical Non-Homologous End Joining (c-NHEJ) pathway is the predominant process in mammals for repairing endogenous, accidental or programmed DNA Double-Strand Breaks. c-NHEJ is regulated by several accessory factors, post-translational modifications, endogenous chemical agents and metabolites. The metabolite inositol-hexaphosphate (IP6) stimulates c-NHEJ by interacting with the Ku70–Ku80 heterodimer (Ku). We report cryo-EM structures of apo- and DNA-bound Ku in complex with IP6, at 3.5 Å and 2.74 Å resolutions respectively, and an X-ray crystallography structure of a Ku in complex with DNA and IP6 at 3.7 Å. The Ku-IP6 interaction is mediated predominantly via salt bridges at the interface of the Ku70 and Ku80 subunits. This interaction is distant from the DNA, DNA-PKcs, APLF and PAXX binding sites and in close proximity to XLF binding site. Biophysical experiments show that IP6 binding increases the thermal stability of Ku by 2°C in a DNA-dependent manner, stabilizes Ku on DNA and enhances XLF affinity for Ku. In cells, selected mutagenesis of the IP6 binding pocket reduces both Ku accrual at damaged sites and XLF enrolment in the NHEJ complex, which translate into a lower end-joining efficiency. Thus, this study defines the molecular bases of the IP6 metabolite stimulatory effect on the c-NHEJ repair activity
