RPA and ATR link transcriptional stress to p53

Abstract

The mechanisms by which DNA-damaging agents trigger the induction of the stress response protein p53 are poorly understood but may involve alterations of chromatin structure or blockage of either transcription or replication. Here we show that transcriptionblocking agents can induce phosphorylation of the Ser-15 site of p53 in a replication-independent manner. Furthermore, microinjection of anti-RNA polymerase II antibodies into the nuclei of cells showed that blockage of transcription is sufficient for p53 accumulation even in the absence of DNA damage. This induction of p53 occurs by two independent mechanisms. First, accumulation of p53 is linked to diminished nuclear export of mRNA; and second, inhibition specifically of elongating RNA polymerase II complexes results in the phosphorylation of the Ser-15 site of p53 in a replication protein A (RPA)-and ATM and Rad3-related (ATR)-dependent manner. We propose that this transcription-based stress response involving RPA, ATR, and p53 has evolved as a DNA damage-sensing mechanism to safeguard cells against DNA damage-induced mutagenesis.antibody microinjection ͉ DNA damage response ͉ RNA polymerase II ͉ nuclear export ͉ phosphorylation T he mechanisms by which DNA lesions are detected and how damage-response pathways are activated in cells are not well understood (1, 2). The stress-response kinases ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) link DNA damage to p53 activation by phosphorylating the Ser-15 site of p53 (3-6). Although the ATM kinase is activated after exposure to ionizing radiation (7), the ATR kinase responds to agents that interfere with replication such as UV light and hydroxyurea (6). The tumor suppressor p53 is induced in response to a variety of different agents in all phases of the cell cycle (8,9). By acting as a transcription factor, p53 can induce the expression of gene products involved in DNA repair, cell cycle arrest, or apoptosis (10, 11). It can also regulate DNA repair and apoptosis by transcription-independent mechanisms (12). Under normal conditions, the p53 protein is rapidly targeted for nuclear export and degradation in a process regulated by the MDM2 protein (13). After cellular stress, the MDM2-mediated negative regulation of p53 is abrogated, and p53 proteins accumulate in the cell nucleus. This nuclear accumulation can be accomplished by (i) DNA damage-induced phosphorylation of p53 and MDM2, leading to the interference of the interaction between MDM2 and p53 (14); (ii) inactivation of proteasomes (15); or (iii) inhibition of the nuclear export machinery (13,16).DNA lesions that block RNA polymerase II induce the recruitment of transcription-coupled repair (TCR) and chromatin remodeling factors to recover RNA synthesis (17)(18)(19). Cells defective in TCR induce p53 and apoptosis at much lower doses than cells with proficient TCR, suggesting that lesions in the transcribed strand of active genes trigger these responses (9,(20)(21)(22). Although these studies have shown a correlation between blockage of ...

5-Fluorouracil signaling through a calcium–calmodulin-dependent pathway is required for p53 activation and apoptosis in colon carcinoma cells

“…1,4,38 Although DNA lesion represent the most well-documented incident causing activation of p53, recent reports also involve transcriptional stress and ribosome damage in signaling events, leading to specific posttranslational modifications of the tumor suppressor. 5,34 An accumulation of double-strand breaks in response to 5-FU in HCT116 cells is evident as the number of gH2AX foci observed by immunostaining increased over time (data not shown), but our data involving ATM inhibition by KU55933 and Chk2-deficient cells excluded the DNA damage response as a starting point for Ca 2 þ -induced p53 phospho activation. Thereby, we are not arguing that apoptosis proceed irrespectively of the DNA damage response.…”

“…1 In comparison with the generally accepted ATM (kinase ataxia telangiectasia mutated)/ATR (Rad3-related protein)-Chk1/Chk2 (checkpoint kinases 1 and 2, respectively)-p53 signaling pathway, leading to apoptotic cell death in response to severe DNA damage, significantly less has been reported regarding death signaling cascades originating from RNA damage, even though it has been proposed that transcriptional stress also can lead to p53 activation. 5 However, despite the extensive use of 5-FU in the clinic, the relative importance for each triggering point is not clearly established. A plausible explanation for this ambiguity can be that, as 5-FU and its metabolites have the potential to strain several discrete molecular mechanisms, treatment outcome is governed by the tumor cell context, which certainly differs between cell types.…”

Deregulated expression of pro-survival and pro-apoptotic p53-dependent genes upon Elongator deficiency in colon cancer cells

“…that defects in both processes trigger a p53-activating stress response. Regarding transcriptional elongation, previous reports established a link between inhibition of transcription and induction of a p53-dependent cellular response [37,39,40]. Whereas these data were obtained using inhibitors of RNA synthesis such as actinomycin D [39], recent reports based on the generation of knock-out mice, undoubtedly provided further evidence for a link between impaired RNA synthesis and p53 activation.…”

“…Interestingly, the Elongin deficient embryos showed activation of the p53 signalling pathways [41]. The blockage of transcriptional elongation through micro-injection of antibodies that specifically recognized the elongating form of RNA polymerase II resulted in p53 accumulation in the nucleus, even in the absence of any DNA damage [40]. We did not observe a significant p53 accumulation upon Elongator deficiency in HCT116 cells but this is most likely due to the fact that only a limited number of genes are not properly transcribed upon Elongator deficiency [11].…”

The role of topoisomerases and RNA transcription in the action of the antitumour benzonaphthyridine derivative SN 28049

“…The role that perturbations in transcription play in the cytotoxicity of topo targeting drugs is still unclear. Accumulation of p53 and the stimulation of p53-dependent apoptosis following inhibition of transcription has been reported [36][37][38] demonstrating that disruption of transcription can lead to cytotoxic events. Recently Derheimer et al (2007) [38] have reported that p53 is strongly induced in human diploid fibroblasts when transcription is blocked, even in the absence of DNA damage, indicating the presence of a response pathway to transcriptional blockage.…”