ATM and KAP1 are required for nonhomologous end-joining in transcriptionally active chromatin

摘要: The effect of ATM signaling on nonhomologous end joining (NHEJ) was investigated using a novel, chromosomally-integrated, viral vector that allows for inducing tandem I-SceI-mediated DNA double strand breaks (DSBs). The DSBs can then be analyzed for NHEJ repair events by fluorescence- and PCR-based methods. Using highly specific kinase inhibitors and this repair system, we show that inhibiting ATM reduces NHEJ by 80% in human U87 glioma cells. PCR products that span the repaired DSBs were analyzed by cleavage with a restriction enzyme that exclusively cuts the DNA repaired by high-fidelity repair. This analysis showed that the extent of high-fidelity repair was reduced by 40% when the ATM kinase was inhibited. KAP1 is a DNA damage-induced phosphorylation target of ATM that is linked to the modulation of chromatin structure. The ATM kinase inhibitor used in our studies blocks radiation …