A TIMELESS-SIRT1-Ku70 complex confers radioresistance to cancer cells by promoting Ku heterodimer assembly

Abstract The Ku heterodimer, composed of Ku70 and Ku80 subunits, is essential for non-homologous end joining (NHEJ). However, the precise mechanisms underlying its assembly and activation remain unclear. Here, we uncovered a novel mechanism for the formation of Ku heterodimer by Ku70 deacetylation. Mechanistically, TIMELESS, as a scaffold protein, bridges SIRT1 and Ku70 to assemble the TIMELESS-SIRT1-Ku70 (TSK) complex. In the presence of TIMELESS, the interaction between SIRT1 and Ku70 was remarkably increased following ionizing radiation (IR), thereby enabling SIRT1-mediated deacetylation of Ku70 at K265 and K331 and promoting the Ku heterodimer formation. The assembly of Ku heterodimer is a prerequisite for initiating and enhancing NHEJ repair efficiency. Therefore, depletion of TIMELESS markedly reduced the Ku heterodimer assembly and impaired NHEJ efficiency. Importantly, targeting TIMELESS sensitizes cancer cells and xenograft mouse models to radiation, highlighting its potential as a therapeutic target for improving radiotherapy outcomes.