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 transcription-blocking 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.

Fig. 1.

Replication-independent phosphorylation of the Ser-15 site of p53 after DNA damage. (A) Diploid human fibroblasts were grown to confluence and then serum-starved for 48 h (0.1% FBS) before being treated with UV light (10 J/m²) and analyzed 6 h later for Ser-15 phosphorylation of p53. (B and C) Cells grown as described above were treated with UV light (10 J/m²), actinomycin D (B), or HMT+UVA (C) and collected 2, 4, or 6 h later, and Ser-15 phosphorylation of p53 was analyzed by using Western blotting. We used β-actin or Coomassie blue staining of total proteins as loading controls. (D) Asynchronously growing diploid fibroblasts were pretreated for 15 min with BrdU to label replicating cells at the time of treatment. The cells were then mock-treated (control) or treated with HMT+UVA. Cells were collected 2 h later, and the induction of Ser-15 phosphorylation of p53 was analyzed in S phase cells (BrdU+, above the line) and in non-S phase cells (BrdU−, under the line). The percentages of Ser-15 phosphorylation-positive cells are indicated for each group.

Fig. 2.

Microinjection of anti-RNA polymerase II antibodies in the nucleus inhibits transcription in human fibroblasts and induces p53 accumulation and phosphorylation. (A) Drawing illustrating what stage in the transcription cycle the different antibodies are expected to abrogate. (B) Human fibroblasts were microinjected in the nucleus with IgG or one of the anti-RNA polymerase II (pol II) antibodies, incubated for 3 h at 37°C, and remicroinjected with the CMV-EGFP-Luc expression vector. After a 1-h incubation at 37°C, the cells were fixed, and the expression of GFP was monitored by immunocytochemistry. White arrows indicate microinjected cells. (C) Human fibroblasts were microinjected in the nucleus with IgG or one of the anti-RNA polymerase II antibodies and incubated for 4 h at 37°C. After fixation, p53 levels were determined by immunocytochemistry. White arrows indicate microinjected cells. (D) Analysis of the percentage of cells in C expressing elevated levels of nuclear p53. (E) Cells were microinjected as in C, but levels of Ser-15-phosphorylated p53 were determined with phospho-specific antibodies. White arrows indicate microinjected cells. (F) Analysis of the percentage of cells in E expressing elevated nuclear levels of Ser-15-phosphorylated p53. The bars in D and F represent the average number of positive cells from five different experiments with 20 microinjected cells per experiment, with error bars representing the SD.

Fig. 3.

Inhibition of mRNA export induces nuclear accumulation of p53 without concomitant phosphorylation of the Ser-15 site. (A) Cells were microinjected with rabbit IgG or anti-TAP antibodies and incubated at 37°C for 4 h followed by fixation and immunofluorescence staining of poly(A) RNA by using biotinylated poly(dT) and Texas red-conjugated streptavidin. White arrows indicate microinjected cells. (B) Human fibroblasts were microinjected in the nucleus with anti-TAP antibodies, incubated for 3 h at 37°C, remicroinjected with the CMV-EGFP-Luc expression vector, and then incubated for 1 h at 37°C. White arrows indicate the microinjected cells. Cells were microinjected with anti-TAP and incubated at 37°C for 4 h followed by fixation and immunocytochemistry for p53 (C) and Ser-15-phosphorylated p53 (D). White arrows indicate the microinjected cells, and 20 J/m² UV light was used as a positive control of Ser-15 phosphorylation in D. (E and F) Analysis of the level of nuclear p53 (E) and Ser-15-phosphorylated p53 (F) in cells microinjected as in C and D, respectively. The bars represent the average number of positive cells from five different experiments with 20 microinjected cells per experiment. (G) Cells were microinjected with a DNA construct expressing a dominant negative Nup160 protein, and 6 h later the cells were fixed and stained. The white arrow indicates the microinjected cell.

Fig. 4.

Induction of the Ser-15 site of p53 after blockage of transcription elongation is mediated by RPA and ATR. (A) (Left) Lymphocytes from an individual affected by the Seckel syndrome (s/s) express very little ATR protein compared with heterozygote cells from a parent (+/s). (Right) (+/s) and (s/s) cells were mock-treated or treated with HMT+UVA. Cells were harvested 6 h later, and the levels of total p53 (Top) and Ser-15-phosphorylated p53 (Middle) were determined by Western blotting. Coomassie blue staining was used to evaluate protein transfer to the membrane. (B) (+/+) and (s/s) cells were irradiated with 10 J/m² and incubated for different periods of time before analysis of Ser-15 phosphorylation of p53. We used β-actin as a loading control. (C) (+/+) and (s/s) cells were treated with actinomycin D for 2 or 4 h before analysis of Ser-15 phosphorylation of p53 was performed with Western blotting. We used β-actin as a loading control. (D) Human fibroblasts were microinjected in the nucleus with anti-ATR antibodies and incubated for 1 h before being irradiated with 20 J/m² UV light (254 nm). After 2 h of incubation at 37°C, the cells were fixed and stained. (E) Human fibroblasts were microinjected with either N20 antibodies alone or with both N20 and anti-ATR antibodies. After incubation for 4 h at 37°C, the cells were fixed and stained. (F) Analysis of the percentage of cells with elevated Ser-15 phosphorylation after comicroinjection with N20 and anti-ATR or H5 and anti-RPA. Because the anti-ATR antibodies are rabbit, they had to be comicroinjected with the rabbit N20 antibodies so that Ser-15 phosphorylation could be detected with mouse antibodies. Conversely, mouse anti-RPA had to be microinjected with mouse H5 antibodies so that Ser-15 phosphorylation could be detected with rabbit antibodies. Bars represent the average of at least five different experiments with 20 injected cells per experiment. The error bars represent SD. The asterisk indicates that the data were taken from Fig. 2F. (G) Ataxia telangiectasia fibroblasts were microinjected with the H5 antibody, and 4 h later the cells were fixed and stained for the presence of H5 antibody, Ser-15 phosphorylation, and DNA. This image is representative of at least 10 injected cells that all stained positive for p53.

Fig. 5.

A model of the transcription stress response leading to the induction of p53 by two different mechanisms. First, inhibition of transcription elongation induces a transcription stress response resulting in phosphorylation of the Ser-15 site of p53 in an RPA- and ATR-mediated manner. Second, loss of mRNA synthesis results in diminished amounts of mRNA available to be exported out of the nucleus (mRNA export also inhibited by anti-TAP antibody microinjection) leading to attenuated nuclear export of p53.