DNA-driven dimerization of Ku70/Ku80 regulates the synapsis of double-strand breaks by APLF

Abstract Repair of double-strand breaks via non-homologous end joining requires the synapsis of DNA ends. APLF is emerging as an important repair factor, capable of forming synaptic bridges in the presence of the Ku70/Ku80 heterodimer. However, only a single one-to-one interaction between Ku70/Ku80 and APLF has been reported to date, which is fundamentally insufficient for synapsis. Moreover, it remains unclear how APLF-mediated bridging of Ku70/Ku80 heterodimers is prevented in the absence of double-strand breaks. Using a combination of single-molecule techniques, including Mass Photometry, Atomic Force Microscopy, and Magnetic and Optical Tweezers, we demonstrate that APLF-mediated synapsis requires threading of at least two Ku70/Ku80 heterodimers per DNA end. We show that this requirement stems from the need for a simultaneous interaction of two poly(ADP-ribose) binding Zinc Fingers in APLF with threaded poly(ADP-ribosyl)ated Ku70/Ku80s on a single DNA. We further provide evidence that long non-coding RNA NIHCOLE cooperatively binds up to four Ku70/Ku80, consistent with a synapsis efficiency enhancement mechanism based on the bridging of two Kus70Ku80s bound on both sides of the break. Our results propose a model for the physiological significance of Ku70/Ku80 threading and a regulatory mechanism based on multivalency that restricts APLF-mediated bridging to DNA-bound Ku70/Ku80.