DNA damage-induced ATM- and Rad-3-related (ATR) kinase activation in non-replicating cells is regulated by the XPB subunit of transcription factor IIH (TFIIH)

Michael G Kemp

doi:10.1074/jbc.m117.788406

Abstract

The role of the DNA damage response protein kinase ataxia telangiectasia-mutated (ATM)- and Rad-3-related (ATR) in the cellular response to DNA damage during the replicative phase of the cell cycle has been extensively studied. However, little is known about ATR kinase function in cells that are not actively replicating DNA and that constitute most cells in the human body. Using small-molecule inhibitors of ATR kinase and overexpression of a kinase-inactive form of the enzyme, I show here that ATR promotes cell death in non-replicating/non-cycling cultured human cells exposed to N-acetoxy-2-acetylaminofluorene (NA-AAF), which generates bulky DNA adducts that block RNA polymerase movement. Immunoblot analyses of soluble protein extracts revealed that ATR and other cellular proteins containing SQ motifs become rapidly and robustly phosphorylated in non-cycling cells exposed to NA-AAF in a manner largely dependent on ATR kinase activity but independent of the essential nucleotide excision repair factor XPA. Although the topoisomerase I inhibitor camptothecin also activated ATR in non-cycling cells, other transcription inhibitors that do not directly damage DNA failed to do so. Interestingly, genetic and pharmacological inhibition of the XPB subunit of transcription factor IIH prevented the accumulation of the single-stranded DNA binding protein replication protein A (RPA) on damaged chromatin and severely abrogated ATR signaling in response to NA-AAF and camptothecin. Together, these results reveal a previously unknown role for transcription factor IIH in ATR kinase activation in non-replicating, non-cycling cells.

Highlights

As one of the major DNA damage response signaling kinases in mammalian cells, the ataxia telangiectasia-mutated (ATM)- and Rad3-related (ATR)2 kinase is primarily thought to respond to DNA polymerase stalling and
The non-cycling cells were treated with a small-molecule ATR inhibitor for 30 min prior to treatment with the carcinogenic UV mimetic NA-AAF, which was employed as a model DNA-damaging agent here because it generates bulky adducts on the C8 position of guanines that block RNA polymerase movement when not removed by the nucleotide excision repair system [33,34,35]
The functions of the ATR kinase in promoting cell survival in response to replication stress are well documented [2, 3, 5, 6]

Summary

Edited by Patrick Sung

The role of the DNA damage response protein kinase ataxia telangiectasia-mutated (ATM)- and Rad-3-related (ATR) in the cellular response to DNA damage during the replicative phase of the cell cycle has been extensively studied. Using the autophosphorylation of ATR and the phosphorylation of SQ motif-containing proteins as biochemical markers of ATR kinase activation, I show that ATR is robustly activated in non-cycling cells exposed to DNA-damaging agents, even at levels of DNA damage that do not yield appreciable cell death This mode of ATR kinase signaling appears to require overt DNA damage because general inhibitors of RNA polymerase function during transcription failed to induce a significant response. Characterization of the activation mechanism of ATR in non-cycling cells unexpectedly revealed a major role for the XPB DNA translocase subunit of transcription factor IIH (TFIIH) in ATR signaling This phenotype was correlated with failure to properly load the single-stranded DNA-binding protein RPA on damaged chromatin. Given that the majority of cells in the body are in a quiescent or non-replicating state, these findings have important implications for understanding the physiology of ATR-dependent DNA damage signaling responses in vivo

Results

Discussion

Cell lines

Chemicals and reagents

RNA interference