Repair of a minimal DNA double‐strand break by NHEJ requires DNA‐PKcs and is controlled by the ATM/ATR checkpoint

Mammalian cells primarily rejoin DNA double‐strand breaks (DSBs) by the non‐homologous end‐joining (NHEJ) pathway. The joining of the broken DNA ends appears directly without template and accuracy is ensured by the NHEJ factors that are under ATM/ATR regulated checkpoint control. In the current study we report the engineering of a mono‐specific DNA damaging agent. This was used to study the molecular requirements for the repair of the least complex DSB in vivo. Single‐chain PvuII restriction enzymes fused to protein delivery sequences transduce cells efficiently and induce blunt end DSBs in vivo. We demonstrate that beside XRCC4/LigaseIV and KU, the DNA‐PK catalytic subunit (DNA‐PKcs) is also essential for the joining of this low complex DSB in vivo. The appearance of blunt end 3′‐hydroxyl and 5′‐phosphate DNA DSBs induces a significantly higher frequency of anaphase bridges in cells that do not contain functional DNA‐PKcs, suggesting an absolute requirement for DNA‐PKcs in the control of chromosomal stability during end joining. Moreover, these minimal blunt end DSBs are sufficient to induce a p53 and ATM/ATR checkpoint function.