Regulation of ATR activity by the RNA polymerase II phosphatase PNUTS-PP1

Ataxia telangiectasia mutated and Rad3-related (ATR) kinase is a key factor activated by DNA damage and replication stress. Here, we show that ATR signaling is increased in human cells after depletion of the RNAPII phosphatase PNUTS-PP1, which dephosphorylates RNAPII on Ser 5 of its carboxy-terminal domain (CTD) (pRNAPII S5). Increased ATR signaling was observed in the presence and absence of ionizing radiation or replication stress and even in G1 phase after depletion of PNUTS. Vice versa, ATR signaling was reduced, in a PNUTS dependent manner, after inhibition of the CDK7 kinase mediating pRNAPII S5. Furthermore, CDC73, a well-known RNAPII-CTD binding protein, was required for the high ATR signaling after depletion of PNUTS and co-immunoprecipitated with RNAPII and ATR. These results suggest a novel pathway involving RNAPII, PNUTS-PP1 and CDC73 in ATR signaling and give new insight into the diverse functions of ATR.