The mutagenic potential of non-homologous end joining in the absence of the NHEJ core factors Ku70/80, DNA-PKcs and XRCC4-LigIV

Non-homologous end joining (NHEJ), the major pathway of double-strand break (DSB) repair in mammalian cells, comprises two subpathways: one that requires the three core factors Ku70/80, DNA-PKcs and XRCC4/LigIV (DNA-PK-dependent NHEJ) and the other that is independent of these factors. Using a cell-free NHEJ assay, we have investigated the ability of three Chinese hamster ovary (CHO) mutants deficient in Ku80 (xrs6), DNA-PKcs (XR-C1) and XRCC4 (XR-1) in comparison with CHO-K1 wild-type cells to rejoin non-compatible DSB ends. Both NHEJ efficiency and fidelity are strongly reduced in the mutants with xrs6 and XR-1 exhibiting the strongest reduction and XR-C1 displaying a phenotype intermediate between the wild-type and the other two mutants indicating a non-essential but facilitating role of DNA-PKcs in NHEJ. The decrease in fidelity in the mutants is expressed by an increase of deletion junctions formed at microhomologies (μhom) near the DSB (microhomology-mediated non-homologous end joining: μhomNHEJ). Using a novel μhomNHEJ assay, we show that μhom regions of 6-10 bp that are located directly at the DSB termini strongly enhance the mutagenic μhomNHEJ reaction even in the wild type. Due to its error proneness, DNA-PK-independent μhomNHEJ may actively promote genome instability. It will, therefore, be of increasing importance to examine NHEJ fidelity in the context with tumorigenesis and cellular senescence for which we here provide two efficient and reliable tools.