Cryo-EM structures of NHEJ assemblies with nucleosomes
- Chloe Hall
- Philippe Frit
- Antonia Kefala-Stavridi
- Amandine Pelletier
- Steven W. Hardwick
- Himani Amin
- Matthew K. Bilyard
- Taiana Maia De Oliviera
- Ammarah Tariq
- Sayma Zahid
- Dimitri Y. Chirgadze
- Shankar Balasubramanian
- Katheryn Meek
- Virginie Ropars
- Jean-Baptiste Charbonnier
- Mauro Modesti
- Patrick Calsou
- Sébastien Britton
- Tom L. Blundell
- Thomas Schalch
- Amanda K. Chaplin
2025-12-24
DNA double-strand breaks (DSBs) are highly deleterious lesions that can trigger cell death or carcinogenesis if unrepaired or misrepaired. In mammals, most DSBs are repaired by non-homologous end joining (NHEJ), which begins when Ku70/80 binds DNA ends and recruits DNA-PKcs to form the DNA-PK holoenzyme. Although recent cryo-EM studies have resolved several NHEJ assemblies, how these factors access DSBs within nucleosomes remains unclear. Here, we present cryo-EM structures of human Ku70/80 and DNA-PK bound to nucleosomes. Ku70/80 binds the DNA end and bends it away from the nucleosome core, while the Ku70 C-terminal SAP domain makes an additional, specific DNA contact. Our DNA-PK–nucleosome structure further reveals the opening of the Ku80 vWA domain, and we show that non-hydrolysable ATP promotes synapsis by stabilising the Ku80-mediated DNA-PK dimer. These structures reveal a model for DSB recognition on nucleosomal DNA and provide insights relevant to targeting NHEJ in cancer therapy.