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)

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.

Keywords: DNA damage; DNA damage response; DNA repair; RNA polymerase; apoptosis; cell cycle; cell signaling; genomic instability; transcription; transcription factor.

Figure 1.

Pharmacological and genetic inhibition of ATR kinase protects non-replicating cells from the lethal effects of the UV mimetic NA-AAF. A, cycling and non-cycling HaCaT cells were pulsed with 10 μg/ml BrdU for 15 min. Genomic DNA was then purified and analyzed by immunodot blotting with the indicated antibodies. The graph shows the relative level of BrdU incorporation into genomic DNA (normalized to cycling cells) from three independent experiments. B, non-cycling HaCaT cells were treated with the indicated concentration of the ATR inhibitor VE-821 for 30 min prior to treatment with 15 μm NA-AAF. Cells were stained with crystal violet 24 h later to determine relative survival. C, cells were treated with the indicated concentration of AZD6738 and analyzed as described in B. D, cells were treated with caffeine and analyzed as described in B. E, non-cycling U2OS cells containing either a WT or KD FLAG-tagged ATR transgene under the control of a tetracycline-inducible promoter were left untreated or treated with 1 μg/ml tetracycline for 48 h before analysis by immunoblotting. F, non-cycling U2OS cells containing the FLAG-ATR-KD transgene were left untreated (− TET, no tetracycline) or treated with tetracycline (+ TET) for 48 h before exposure to the indicated concentration of NA-AAF. After an additional 48 h, cells were stained with crystal violet to determine relative survival. G, non-cycling U2OS cells induced to express the indicated form of ATR were treated with NA-AAF as in E to determine relative cell survival. *, p < 0.05; indicating a significant difference in survival between the two treatments or cell lines.

Figure 2.

ATR autophosphorylation on Thr-1989 is a marker of ATR activation in non-cycling cells exposed to the UV mimetic NA-AAF. A, cycling and non-cycling HaCaT cells were treated with 20 μm NA-AAF for 1 h. Cell lysates were analyzed by immunoblotting with antibodies targeting the indicated proteins and phosphorylated residues (P). Quantitation of ATR autophosphorylation (average and standard error) from three independent experiments is provided below a representative immunoblot. The phospho-ATR signal was normalized to the total ATR signal for each sample, which was then compared with the NA-AAF–treated cycling cell sample in each experiment (set to an arbitrary value of 100). B, non-cycling HaCaT cells were pretreated with DMSO or 10 μm VE-821 (an ATR inhibitor) for 30 min prior to treatment with 10 μm NA-AAF. Cells were harvested at the indicated time point and analyzed by immunoblotting. C, quantitation of results from at least two independent experiments performed as in B. D, U2OS cells induced with tetracycline for 2 days to express either wild-type or kinase-dead forms of ATR were left untreated or treated with 60 μm NA-AAF for 4 h, and then cell lysates were analyzed by immunoblotting. Quantitation of three independent experiments is provided below the representative immunoblot data. The phospho-ATR signal was normalized to total ATR, and this ratio was set to an arbitrary value of 100 for NA-AAF–treated cells expressing WT ATR. All other samples were compared with this value. *, p < 0.05; indicating a significant difference in NA-AAF–induced ATR phosphorylation in WT and KD cells.

Figure 3.

Analysis of DNA damage–induced protein phosphorylation events in non-cycling cells reveals a major role for the ATR kinase. A, cycling and non-cycling HaCaT cells left untreated or treated for 1 h with 20 μm NA-AAF before harvesting and analysis by immunoblotting with the indicated antibodies, which included an antibody mixture that recognizes phosphorylated SQ motifs (SQ-P) common to ATR and ATM kinase substrates. The graph shows the quantitation of three independent experiments. The total phosphoprotein signals on the blots were normalized first to the Ponceau S stain and then to the NA-AAF–treated cycling cell sample, which was set to an arbitrary value of 100. *, p < 0.05; indicating a significant difference in protein phosphorylation between cycling and non-cycling cells. B, non-cycling HaCaT cells were treated with the indicated concentration of NA-AAF. Cells were harvested 1 h later and analyzed by immunoblotting. C, non-cycling cells were treated with 10 μm NA-AAF, and cells were harvested at the indicated time points and analyzed by immunoblotting. D, cells were treated with the indicated DNA damage response kinase inhibitor for 30 min prior to addition of 20 μm NA-AAF. Cells were harvested 2 h later and analyzed by immunoblotting. The graph shows the relative SQ motif phosphorylation (normalized to the DMSO control) from three independent experiments. *, p < 0.05; indicating a significant difference in SQ motif phosphorylation between DMSO- and ATR inhibitor/ATM inhibitor–treated cells. E, U2OS cells expressing either wild-type or kinase-dead ATR were left untreated or treated with 20 μm NA-AAF for 2 h, and then cell lysates were analyzed by immunoblotting. The graph quantifies the SQ motif phosphorylation from three independent experiments, which was significantly reduced (*, p < 0.05) in NA-AAF-treated cells expressing the kinase-dead form of ATR.

Figure 4.

Depletion of the nucleotide excision repair factor XPA does not prevent ATR kinase signaling in non-cycling cells exposed to NA-AAF. A, HaCaT cells transduced with either a control or XPA shRNA–expressing vector were analyzed by Western blotting to verify knockdown of the XPA protein. B, control or XPA shRNA-expressing HaCaT cells maintained in a non-cycling state were exposed to the indicated concentration of NA-AAF and then stained with crystal violet to validate that the loss of XPA increases the sensitivity of the cells to NA-AAF. *, p < 0.01; indicating a significant difference in survival between the two cell lines at the indicated doses of NA-AAF. C, control and XPA shRNA–expressing HaCaT cells were left untreated or treated with 20 μm NA-AAF and harvested for immunoblot analysis 2 h later. SQ-P, phosphorylated SQ motif. D, quantitation of three independent experiments performed as in C. Phosphoprotein signals were normalized to the NA-AAF–treated control cell sample, which was set to an arbitrary value of 100.

Figure 5.

Direct DNA damage, but not general transcription stress, leads to robust activation of ATR kinase signaling in non-cycling cells. A, non-cycling HaCaT cells were treated with DMSO or the ATR inhibitor VE-821 before exposure to 2.5 μm CPT, 100 μm DRB, 300 nm TPL, 20 μm NA-AAF, or 30 ng/ml ActD. Cells were harvested 4 h later and analyzed by immunoblotting. B, quantitation of ATR and p53 phosphorylation from four independent experiments performed as in A. Signals were compared with the NA-AAF–treated samples, which were set to an arbitrary value of 100. *, p < 0.05; indicating a significant difference in protein phosphorylation between drug-treated and DMSO-treated cells.

Figure 6.

Inhibition TFIIH with triptolide abrogates the bulk of ATR kinase signaling in response to DNA damage in non-cycling cells. A, non-cycling HaCaT cells were pretreated with DMSO or the indicated transcription inhibitor for 30 min prior to exposure to 20 μm NA-AAF. Cells were harvested 2 h later and analyzed by immunoblotting. B, quantitation of ATR, p53, and ATM/ATR substrate (SQ-P) phosphorylation from four independent experiments performed as in A. The phosphoprotein samples from cells treated with DMSO + NA-AAF were set to an arbitrary value of 100, and all other samples were compared with this value. C, non-cycling HaCaT cells were treated and analyzed as in A and B, except that cells were treated with camptothecin instead of NA-AAF. *, p < 0.05l; indicating a significant difference in protein phosphorylation between drug-treated and DMSO-treated cells. D, cells were treated as in A, except that cells were harvested 1 h after NA-AAF administration and then fractionated to isolate chromatin-associated proteins. The graph shows the relative level of chromatin-associated RPA70 (normalized to Ponceau staining) from three independent experiments. *, p < 0.05; indicating a significant difference in RPA chromatin level between TPL- and DMSO-treated cells.

Figure 7.

The XPB subunit of TFIIH is required for ATR kinase activation in response to DNA damage in non-cycling cells. A, non-cycling HaCaT cells were pretreated with DMSO, 1 μm TPL, 10 μm SP, or 1 μm THZ1 for 30 min prior to exposure to 15 μm NA-AAF. Cells were harvested 2 h later and analyzed by immunoblotting. The graph shows the quantitation of SQ motif protein phosphorylation (SQ-P) from at least three independent experiments. *, p < 0.05; indicating a significant difference in protein phosphorylation between drug-treated and DMSO-treated cells. B, HaCaT cells were transfected with control or XPB siRNAs prior to treatment with NA-AAF as in A. Cell lysates were analyzed by immunoblotting to verify XPB knockdown and SQ motif phosphorylation. The graph shows the relative level of SQ motif–containing protein phosphorylation from three independent experiments. *, p < 0.05; indicating a significant difference between control and XPB siRNA–transfected cells.