Cryo-EM structures of NHEJ assemblies with nucleosomes

Area of Science:

• Molecular Biology
• Structural Biology
• Genetics

Background:

• DNA double-strand breaks (DSBs) are dangerous DNA lesions that can lead to cell death or cancer if not repaired correctly.
• Non-homologous end joining (NHEJ) is the primary pathway for repairing DSBs in mammals, initiated by Ku70/80 binding to DNA ends and recruiting DNA-PKcs.
• The precise mechanism by which NHEJ factors access DSBs located within nucleosomes has remained elusive.

Purpose of the Study:

• To elucidate the structural basis of how Ku70/80 and DNA-PK interact with nucleosomes containing DSBs.
• To provide a molecular model for DSB recognition within the context of chromatin.
• To identify potential targets within the NHEJ pathway for cancer therapy.

Main Methods:

• Utilized cryo-electron microscopy (cryo-EM) to determine high-resolution structures of human Ku70/80 and DNA-PK bound to nucleosomes.
• Performed biochemical assays to investigate the role of ATP in DNA-PK complex formation and synapsis.

Main Results:

• Presented cryo-EM structures detailing the binding of Ku70/80 and DNA-PK to nucleosomal DNA.
• Demonstrated that Ku70/80 binds DSB ends, bending the DNA away from the nucleosome core, with the Ku70 C-terminal SAP domain forming an additional DNA contact.
• Showed that non-hydrolysable ATP stabilizes the DNA-PK dimer via Ku80, facilitating synapsis and revealing the opening of the Ku80 vWA domain.

Conclusions:

• Proposed a structural model for how NHEJ factors recognize and initiate repair of DSBs within nucleosomes.
• The findings offer critical insights into the structural dynamics of DNA repair machinery at chromatinized DNA breaks.
• These structural insights are relevant for developing novel cancer therapies that target the NHEJ pathway.