ATR kinase; attenuation; fork restart; PPM1D; replication stress
In eukaryotes, in response to replication stress, DNA damage response kinase, ATR is activated, whose signalling abrogationleads to cell lethality due to aberrant fork remodelling and excessive origin firing. Here we report that inhibition ofATR kinase activity specifically during replication stress recovery results in persistent ATR signalling, evidenced by thepresence of ATR-dependent phosphorylation marks (cH2AX, pChk1 and pRad17) and delayed cell cycle re-entry. Further,such disruption of ATR signalling attenuation leads to double-strand breaks, fork collapse and thereby ‘replicationcatastrophe’. PPM1D phosphatase, a nucleolar localized protein, relocates to chromatin during replication stress and revertsback to nucleolus following stress recovery, under the control of ATR kinase action. Inhibition of ATR kinase activity,specifically during post replication stress, triggers dislodging of the chromatin-bound PPM1D from nucleus to cytoplasmfollowed by its degradation, thereby leading to persistence of activated ATR marks in the nuclei. Chemical inhibition ofPPM1D activity or SiRNA mediated depletion of the protein during post replication stress recovery ‘phenocopies’ ATRkinase inhibition by failing to attenuate ATR signalling. Collectively, our observations suggest a novel role of ATR kinasein mediating its own signal attenuation via PPM1D recruitment to chromatin as an essential mechanism for restarting thestalled forks, cell-cycle re-entry and cellular recovery from replication stress.