DNA-PK driven nucleosome unwrapping enables NHEJ in chromatin

Abstract DNA double-strand breaks, one of the most cytotoxic forms of DNA damage, are primarily repaired by non-homologous end joining (NHEJ) in human cells. NHEJ is initiated by the Ku70/80 heterodimer (Ku) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs), followed by factors that bridge and ligate DNA ends. Ku and DNA-PKcs require ~28 base pairs of free duplex DNA, which is often unavailable in chromatin with nucleosomes acting as barriers. The role of DNA-PKcs remains unclear, as previous in vitro studies mainly used naked DNA. Here, in vitro ligation assays show that DNA-PKcs promotes NHEJ on nucleosomes with limited DNA accessibility. Cryo-EM structures of nucleosome-bound Ku and DNA-PKcs reveal that Ku encounters nucleosomal barriers and overcomes them with DNA-PKcs. Distinct structural states support a stepwise model of DNA-PK progressive translocating along nucleosomal DNA, highlighting DNA-PKcs function in chromatin-associated NHEJ in vivo.