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[Cancer Research 65, 10810-10821, December 1, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Evidence for the Direct Binding of Phosphorylated p53 to Sites of DNA Breaks In vivo

Shahnaz T. Al Rashid1,2, Graham Dellaire4, Andrew Cuddihy1, Farid Jalali1, Mita Vaid5, Carla Coackley1, Melvyn Folkard5, Yang Xu6, Benjamin P.C. Chen7, David J. Chen7, Lothar Lilge1,2, Kevin M. Prise5, David P. Bazett Jones4 and Robert G. Bristow1,2,3

1 Ontario Cancer Institute Princess Margaret Hospital, University Health Network; Departments of 2 Medical Biophysics and 3 Radiation Oncology, University of Toronto; 4 Programme in Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada; 5 Gray Cancer Institute, Mount Vernon Hospital, Northwood, Middlesex, United Kingdom; 6 Division of Biological Sciences, University of California, San Diego, La Jolla, California; and 7 Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas

Requests for reprints: Robert G. Bristow, Experimental Therapeutics, Ontario Cancer Institute/Princess Margaret Hospital, Room 5-923, 610 University Avenue, Toronto, Ontario, Canada M5G2M9. Phone: 416-946-2129; Fax: 416-946-4586; E-mail: rob.bristow{at}rmp.uhn.on.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Despite a clear link between ataxia-telangiectasia mutated (ATM)–dependent phosphorylation of p53 and cell cycle checkpoint control, the intracellular biology and subcellular localization of p53 phosphoforms during the initial sensing of DNA damage is poorly understood. Using G0-G1 confluent primary human diploid fibroblast cultures, we show that endogenous p53, phosphorylated at Ser15 (p53Ser15), accumulates as discrete, dose-dependent and chromatin-bound foci within 30 minutes following induction of DNA breaks or DNA base damage. This biologically distinct subpool of p53Ser15 is ATM dependent and resistant to 26S-proteasomal degradation. p53Ser15 colocalizes and coimmunoprecipitates with {gamma}-H2AX with kinetics similar to that of biochemical DNA double-strand break (DNA-dsb) rejoining. Subnuclear microbeam irradiation studies confirm p53Ser15 is recruited to sites of DNA damage containing {gamma}-H2AX, ATMSer1981, and DNA-PKcsThr2609 in vivo. Furthermore, studies using isogenic human and murine cells, which express Ser15 or Ser18 phosphomutant proteins, respectively, show defective nuclear foci formation, decreased induction of p21WAF, decreased {gamma}-H2AX association, and altered DNA-dsb kinetics following DNA damage. Our results suggest a unique biology for this p53 phosphoform in the initial steps of DNA damage signaling and implicates ATM-p53 chromatin-based interactions as mediators of cell cycle checkpoint control and DNA repair to prevent carcinogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The cellular stress response to DNA damage requires strict coordination between cell cycle checkpoint control and DNA repair. In response to DNA double-strand breaks (DNA-dsb), the ataxia-telangiectasia mutated (ATM), DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and hSMG-1 kinases, all members of the phosphatidylinositol 3-kinase–related kinase (PI3KK) family, have been shown to redundantly phosphorylate substrates, including the DNA-dsb sensor histone protein, {gamma}-H2AX, and the p53 tumor suppressor protein (1–4). The presence of DNA-dsbs activates an initial autophosphorylation of ATM, resulting in ATM monomers phosphorylated at Ser1981 (ATMSer1981). This early activation of ATM is facilitated by the MRE11/RAD50/NBS1 (MRN) complex and protein phosphatases 2A and 5 (PP2A and PP5) and hPTIP (5–8). Similarly, DNA-PKcs, involved in the nonhomologous end-joining (NHEJ) DNA-dsb repair pathway, is also activated by PP5 following DNA breaks and becomes autophosphorylated at its Thr2609 residue (DNA-PKcsThr2609; refs. 3, 9, 10). Specifically, the NH2 terminus protein interaction domain of ATM directly phosphorylates p53 at Ser15 residue and CHK2 at Thr68 residue (11). The stabilization and activation of p53 induces transcription of p21WAF, an inhibitor of the cyclin E/cyclin-dependent kinase 2 complex and RB phosphorylation leading to cell arrest at the G1-S transition. ATM-mediated p53 phosphorylation also abrogates the interaction between p53 and MDM2 (an E3 ubiquitin ligase) thus inhibiting ubiquitination of p53 and its degradation by the 26S proteasome. Altogether, ATM-mediated phosphorylation of p53 and CHK2 leads to a series of G1 and G2 cell cycle checkpoints that together act in preventing genomic instability following DNA damage (12).

An outstanding question still remains as to what function(s) are associated with p53 phosphoforms that are activated during the initial sensing and transduction of irradiation-induced DNA damage by ATM. Recent reports of clustered interdependence between select p53 phosphorylation sites suggest that certain p53 phosphoforms may have unique biology pertaining to DNA damage sensing or repair. For example, Ser15 residue phosphorylation occurs within 15 minutes of exposure to irradiation leading to a subsequent clustered phosphorylation of NH2-terminal residues Thr18, Ser9, and Ser20 (11). These modifications are not solely linked to p53 protein stabilization (13, 14). O'Hagan et al. have recently reported that Ser15 phosphorylation can be uncoupled from p53 nuclear accumulation, consistent with the concept that other possible biological activities may be associated with phosphorylation of the Ser15 residue (15). The timing, intracellular locale, and exact residues of p53 phosphorylation, dephosphorylation, and acetylation may reflect the level and type of DNA damage following chromatin or nucleolar disruption (11, 13, 16).

ATM can sense changes in global chromatin structure or changes resulting from DNA breaks leading to pan-nuclear phosphorylation of its substrates, including that of p53 (17). Based on live cell imaging of fluorescently tagged proteins, ATM can also phosphorylate its targets (e.g., NBS1) directly at the site of DNA damage. This gives rise to dynamic local protein-chromatin interactions, including CHK2 phosphorylation at the site of DNA damage (18, 19). Individually, ATM and p53 have been shown to bind double-stranded DNA, irradiation-induced DNA-dsbs, and DNA base damage and be cofactors in NHEJ or homologous recombination based on DNA-dsb repair assays in vitro and in vivo (20–24). It is therefore plausible that irradiation-induced p53 or ATM phosphoforms may directly interact with sites of DNA damage and interact with DNA repair proteins during the initial hours of maximal DNA-dsb recognition and repair (24–26).

Intracellular interactions between ATM-associated proteins and DNA repair proteins can now be tracked in situ using immunofluorescence microscopy in which discrete nuclear protein-protein interactions can be visualized following whole-cell or subcellular (e.g., UV microbeam) radiation at sites of DNA damage (8, 17, 27–30). Biochemical and microscopic studies support the phosphorylation of H2AX at Ser139 (i.e., {gamma}-H2AX) as a discrete biomarker of megabase domains containing DNA-dsbs (28). Furthermore, residual {gamma}-H2AX foci at late time points following irradiation (i.e., 24 hours) are thought to represent nonrepaired sites of DNA damage, which correlate with relative radiation cell survival in vitro and in vivo (30, 31). {gamma}-H2AX was also found to be essential for the recruitment of 53BP1, BRCA1, MDC1, and the MRN complex to the site of DNA damage during ATM-mediated phosphorylation (28). Recently, Kang et al. found that {gamma}-H2AX was dispensable for the activation of ATM and p53 responses following DNA damage, and that NBS1, {gamma}-H2AX, and p53 can interact in parallel with ATM to maintain genetic stability (32). These results are supported by the recent work of Bartkova et al. in which increased endogenous activation of DNA damage signaling proteins (e.g., ATMSer1981, CHK2Thr68, {gamma}-H2AX, and p53Ser15) was a biomarker of genetic instability and malignancy as a response to aberrant DNA replication in transformed cells (33). With the development of these phospho-specific antibodies, the phosphorylated p53 isoforms and their intracellular distribution following DNA damage can be studied in relation to total cellular p53 and other signaling and DNA repair proteins.

We hypothesized that p53Ser15 might participate within a larger genome surveillance complex within the first hour after irradiation, similar to that reported for the ATM, DNA-PKcs, {gamma}-H2AX/53BP1, and the MRN complex (8, 10, 29). To test this hypothesis, we used primary human fibroblast strains to determine the expression and subcellular localization of endogenous p53 species following DNA damage. Herein, we report that discrete p53Ser15 nuclear foci are maximally induced by DNA breaks and base damage. These foci form within minutes from a preexisting pool of p53. Using a variety of methodologies, including whole-cell and subnuclear irradiation techniques, we have determined that p53Ser15 coimmunoprecipitates and colocalizes with {gamma}-H2AX and interacting proteins at the site of DNA damage. Our data place the p53Ser15 phosphoform at the site of DNA-dsbs during the initial surveillance of DNA damage and further exemplifies the unique biology of selected p53 phosphoforms during DNA damage signaling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Fibroblast strains and cell culture conditions. Primary human fibroblast strains were obtained from Coriell (Camden, NJ) and cultured as per supplier's instructions: normal diploid fibroblast strains NDF-GM03651 and NDF-GM05757; homozygous ATM–/– strains AT-GM05823 (also known as AT5B1) and AT-GM02052 (24); homozygous NBS–/– strain NBS-GM07166 (34); and the DNA Ligase IV–deficient strain GM016088 (also known as 180BR), which shows a defective rate of DNA-dsb rejoining. Genomic DNA from all strains was isolated and sequenced for p53 to confirm that the NDF, AT, and NBS fibroblast strains expressed two wild-type p53 alleles. Isogenic p53WT/WT and p53–/– HCT116 colon carcinoma cell lines (a gift from Dr. B. Vogelstein, Department of Oncology, John Hopkins University, Baltimore, MD) and the p53WT/WT and p53Ser18Ala/Ser18Ala murine embryo fibroblasts (MEF) have both been previously described (35).

To preclude cell cycle bias in the initial quantification of p53 and {gamma}-H2AX responses, a synchronization protocol was used to obtain density-inhibited, G0-G1 phase cultures as previously described by Rothkamm et al. (12) and Little et al. (34). Flow cytometric analyses confirmed 90% to 95% G1 content in NDF strains and 80% to 85% G1 content in AT, LFS, and NBS strains before use. In selected experiments, asynchronous populations were also used.

Irradiation and chemical induction of DNA damage. G0-G1 phase or asynchronous cultures were exposed to whole-cell ionizing radiation (dose range, 0-20 Gy) using a 137Cs irradiator (MDS Nordion, Ottawa, Ontario, Canada) at ~1 Gy/min (room temperature, aerobic conditions; ref. 36). UV irradiation (20 J/m2) was carried out in PBS using a continuous UV wavelength source (254 nm) as previously described (36). For subnuclear damage experiments, a high-linear energy transfer (LET; 100 keV/µm) helium-3 ion microbeam was used to target specific subnuclear locales within G0-G1 phase cells prestained with 1 µmol/L Hoechst33258 (Molecular Probes, Eugene, OR; method described in detail by Belyakov et al.; ref. 37). In complementary subnuclear targeting experiments, a UV laser microbeam was used as previously described (28, 38). In this case, cells were irradiated with a 390- nm laser using a Laser Scissors Module 390/20 microscopic setup (Cell Robotics, Inc., Albuquerque, NM) at 75% power output (10 pulses/s and maximum rate of 10 µm/s).

For drug treatments, cells were also exposed to the PI3KK inhibitor, wortmannin (23 µg/mL for 1 hour); DNA-dsb-inducing agent, bleomycin (0.06 units/mL for 3 hours); DNA single-strand break (DNA-ssb)–inducing agent, H2O2 (100 µmol/L for 1 hour); DNA base damage–inducing agent, methyl methane sulfonate (MMS; 0.01% or ~1 mmol/L for 1 hour); and DNA cross-linking agent, mitomycin C (MMC; 1 µg/mL for 2 hours). Logarithmically growing cultures were exposed to hydroxyurea (1 mmol/L for 16 hours), an inhibitor of DNA replication (21). G0-G1 phase NDF-GM05757 fibroblasts were also pretreated with 30 µg/mL cyclohexamide at 15 minutes before irradiation to inhibit protein synthesis or 10 µmol/L MG132 (for various times after irradiation) to inhibit the activity of the 26S proteasome. Control cultures incubated with and without fetal bovine serum, 0.1% DMSO, or ethanol carriers served as negative controls for the latter experiments.

Confocal and wide-field immunofluorescence microscopy. Intranuclear staining patterns of endogenous or exogenous protein expression were visualized using immunofluorescence microscopy. Cells cultured in four-chamber slides (Lab-Tek, Nalgene, Rochester, NY) were fixed and permeabilized in 2% paraformaldehyde/0.2% Triton X-100 (pH 8.2) and 0.5% NP40. Fixed cells were then incubated with diluted primary and fluorophore-conjugated secondary [rhodamine red X (red) or FITC (green); Jackson ImmunoResearch, West Grove, PA] antibodies in 3% bovine serum albumin (BSA). Negative staining controls included cells incubated with 3% BSA alone or IgG in 3% BSA. Finally, cells were counterstained for nuclear DNA using 0.1 µg/mL 4',6-diamidino-2-phenylindole (DAPI) before mounting in Vectashield (Vector Labs, Burlingame, CA) for microscopic analyses. Of note, fixation and permeabilization of cultures including using either 4% paraformaldehyde or methanol/acetone gave similar and consistent staining patterns.

To determine whether p53Ser15 foci were soluble or associated with nuclear matrix, RNA or DNA, the G0-G1-synchronized fibroblasts were permeabilized or permeabilized and pretreated with either RNaseA or DNaseI, as previously described by Rubbi and Milner (39). Briefly, cells were washed with TBS [150 mmol/L NaCl, 10 mmol/L Tris, 5 mmol/L MgCl2 (pH 7.4)], twice with TBS-G (TBS, 25% glycerol, 0.5 mmol/L EGTA), and then for three to four minutes in TBS-G-TX (TBS-G, 0.05% Triton X-100). The cells were then either incubated at room temperature for 1 hour in S buffer (soluble proteins are preextracted), or RNaseA in S buffer (200 units/mL; Invitrogen, Carlsbad, CA), or DNaseI in TBS (1 µg/µL, Invitrogen). Following this preextraction/treatment, the cells were processed as indicated above for immunofluorescence staining and detection of the proteins of interest.

Images were captured using a Zeiss LSM510 confocal microscope at a final magnification of x630. The use of 1.8-µm confocal sections allowed for a quantitative comparison of responses for doses up to 20 Gy. Nuclei with three or more foci were designated as foci-positive nuclei as previously described (40). For all experiments, at least 30 to 50 nuclei were scored after controlling for background staining based upon nonirradiated cultures. The final data is presented as the mean of two to six independent experiments with the associated SE. Significant colocalization of nuclear foci was determined by visualization of merged red and green (resulting in yellow) images upon a DAPI background. Colocalization was confirmed by characterization of the fluorescence intensity profiles for given fluorochromes within a defined subnuclear region and confirmed by the calculation and plotting of the Pearson's correlation coefficient (rp; ref. 39). A lack of perfect alignment between channels monitoring different fluorophores results in the coefficient values oscillating around a constant background value. When channels monitoring different fluorophores correlate (or anticorrelate), the coefficients will depart positively (or negatively) from the background value.

Western blot, immunoprecipitation, and cellular fractionation analyses. Immunoprecipitation and Western blot analyses were done using standard protocols as previously described (41). Cytoplasmic, nuclear, and chromatin-bound cellular fractions were isolated using a modified Dignam method (42). Briefly, cells were lysed and incubated for 5 minutes on ice, in cytoplasmic buffer [25 mmol/L KCl, 5 mmol/L MgCl2, 10 mmol/L Tris-HCl (pH 8), 0.5% NP40, 1 mmol/L DTT, 1x protease inhibitors (Complete EDTA-free, Roche, Indianapolis, IN), 1x phosphatase inhibitors (Cocktail Set II, Calbiochem, La Jolla, CA)]. Lysates were centrifuged at 3,000 rpm for 5 minutes, and the supernatant (cytoplasmic fraction) was separated. The nuclear pellet was rinsed thrice with cytoplasmic buffer and resuspended in nuclear buffer [10 mmol/L Tris-HCl (pH 8), 500 mmol/L NaCl, 0.1% NP40, 5 mmol/L EDTA, 1x protease inhibitors (Complete EDTA-free, Roche), 1x phosphatase inhibitors (Cocktail Set II, Calbiochem)]. The nuclei were lysed by vigorous pipetting, vortexed for 5 minutes, and incubated on ice for 15 minutes. The nuclear fraction was centrifuged at 13,000 rpm for 15 minutes to pellet the chromatin, and the supernatant (nuclear fraction) was separated. The chromatin pellet was rinsed thrice with nuclear buffer, resuspended in nuclear buffer, and subjected to 20 pulses of sonication (Branson Sonifier 450, 60 Hz) to shear the DNA. All fractions were then analyzed by Western blot as indicated above.

Antibodies used in this study include p53Ser15 (Ab-3), Ab-7 (pantropic p53), DO-1 (NH2 terminus–specific p53), Ab421 (COOH terminus–specific p53), p21WAF (Ab-1), RAD51 (Ab-1), BRCA1 (Ab-1), and {alpha}-tubulin (Ab-1) from Oncogene Research Products (Cambridge, MA); Ab1801 (NH2 terminus–specific p53) from Novocastra (Norwell, CA); polyclonal Ser6-, Ser9-, Ser15-, Ser20-, Ser37-, Ser46- and Ser392-phosphorylated p53 and monoclonal Ser15-phosphorylated p53 and Ab9282 (pantropic p53) from Cell Signaling (Beverly, MA); polyclonal and monoclonal {gamma}-H2AX from Upstate Biotechnology (Charlottesville, VA); ATMSer1981 from Rockland Immunochemicals (Gilbertsville, PA); RAD50 (2C6) from Novus Biologicals (Littleton, CO); MRE11 from Genetex (San Antonio, TX); actin from Sigma-Aldrich (St. Louis, MO); PML (1B9) from MBL (Woburn, MA); 53BP1, FL393, BP-Aldrich-12 (pantropic p53), and nucleolin/C23 from Santa Cruz Biotechnology (Santa Cruz, CA); green fluorescence protein [also recognizes yellow fluorescent protein (YFP) variant] from BD Biosciences (San Jose, CA); and DNA-PKcsThr2609 as previously described (10).

The specificity of p53 antibodies was confirmed using p53–/– cells (SAOS-2, PC3, and HCT116 p53–/–) in which no detectable p53 and p53Ser15 protein expression was observed following Western blot, immunoprecipitation, and immunostaining analyses. Two polyclonal (rabbit; Oncogene and Cell Signaling) and one monoclonal (mouse; Cell Signaling) p53Ser15-specific antibodies all revealed similar staining patterns before and after irradiation. Thus, all subsequent experiments were done using the polyclonal rabbit Oncogene p53Ser15-specific antibody.

DNA double-strand break rejoining assays. Biochemical DNA-dsb rejoining kinetics for human and murine fibroblasts were determined using the continuous-field gel electrophoresis (CFGE) assay and neutral Comet assay as previously described (36, 41). CFGE assays have been used to accurately quantify DNA-dsb rejoining kinetics (12). Briefly, cells were grown in 60-mm dishes and either irradiated or mock irradiated on ice and then incubated at 37C in fresh medium until lysis at various times of 0 to 24 hours after irradiation (100 and 20 Gy for the CFGE and COMET assays, respectively). For the CFGE assay, samples were loaded into the wells of a 0.8% agarose/0.5x Tris-borate EDTA (TBE) gel before electrophoresis at room temperature for 40 hours at 0.6 V cm–1 in 0.5x TBE buffer. After electrophoresis, gels were stained with 1 µg ml–1 ethidium bromide, destained in deionized water, and imaged using an UV imaging system equipped with a CCD camera and imaging software (LabWorks, UVP, Inc., Upland, CA).

For MEFs in which total cell numbers were limiting, the single-cell neutral Comet assay was used. Single-cell suspensions were mixed with 75 µL of 0.5% low-melting agarose at 37°C and spread on a 1% agarose precoated slide. Slides were then incubated in Proteinase-K solution for 60 minutes at 37°C followed by incubation in ice-cold lysis buffer (2.5 mol/L NaCl, 100 mmol/L EDTA, 10 mmol/L Trizma base, 10% DMSO, 1% Triton-X) overnight. After lysis, the slides were placed in horizontal electrophoresis tanks filled with electrophoresis buffer (1x TBE, pH 8.0) for 20 minutes and then subjected to electrophoresis at 25 V/30 to 45 mA for a further 20 minutes. After electrophoresis, the slides were washed (0.4 mol/L Tris-HCl, pH 7.5) thrice, air-dried, and stained with ethidium bromide (2 µg ml–1) before scoring. The relative amount of fragmented DNA contained within the Comet's tail, compared with the nonfragmented DNA within the Comet head, was determined by fluorescent image analysis (Northern Eclipse software) to determine the normalized tail moment as a measure of residual DNA breaks over time following irradiation.

Yellow fluorescent protein-p53 phosphomutant constructs and transient transfection conditions. The role of the Ser15 residue in mediating foci formation was tested by expressing exogenous YFPs fused to p53WT or p53Ser15Ala (incapable of Ser15 phosphorylation) proteins to track subcellular protein patterns following DNA damage. Briefly, the p2516 plasmid containing the human full-length p53WT/WT cDNA fragment cloned into the pcDNA3.1 backbone vector was used as a template for generating the p53 fusion and mutant constructs (43). The full-length p53 fragment was amplified from p2516 plasmid DNA using the forward and reverse oligonucleotide primers, 5'-TTTTAAGCTTCGATGGAGGAGCCGCAGTCAGA-3' and 5'-TTTTGGATCCTCAGTCTGAGTCAGGCCC-3', respectively. The PCRs were optimized with the Platinum Pfx DNA polymerase (Life Technologies Bethesda Research Laboratories, Frederick, MD) in a Peltier Thermal Cycler (MJ Research, BIO-RAD, Waltham, MA). The amplified fragment was subjected to BamHI and HindIII digestion (New England Biolabs, Ipswich, MA), ligated in-frame, and fused to the COOH terminus of the enhanced YFP gene in the pEYFP-C1 vector (Clontech Laboratories, Inc., Mountain View, CA). Subsequently, the Ser15 site of this clone, YFP-p53, was specifically mutated using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) to generate the YFP-p53Ser15Ala clone using the following primers: Ser15Ala, forward 5'-AGCGTCGAGCCCCCTCTGGCTCAGGAAACATTTTCAGAC-3' and Ser15Ala, reverse 5'-GTCTGAAAATGTTTCCTGAGCCAGAGGGGGCTCGACGCT-3'. All plasmid clones were purified using the CONCERT High Purity Maxiprep System (Life Technologies Bethesda Research Laboratories) and sequenced on both strands to confirm wild-type and site-specific mutated status. Metafectene (Biontex, Munich, Germany) was used to transfect the HCT116 p53WT/WT and HCT116 p53–/– cells with YFP-p53 vectors. All reactions were carried out according to the manufacturer's instructions. Following transfection, cells were fixed and imaged at regular intervals following irradiation using a Zeiss LSM510 confocal microscope.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ionizing radiation induces discrete dose-responsive p53Ser15 nuclear foci from a preexisting p53 protein pool in an ataxia-telangiectasia mutated–dependent manner. To our knowledge, there are no systematic microscopic studies of relative intracellular localization of p53 phosphoforms following DNA damage. Therefore, a panel of p53 pan- and phospho-specific antibodies was initially tested to determine relative p53 phosphorylation and subnuclear localization following whole-cell irradiation (Fig. 1A-D). These included pantropic p53 antibodies that recognized the NH2 terminus (Ab1801 and DO-1), COOH terminus (Ab421), full-length p53 (BP53-12, Ab-7, and FL393), and specific p53 phosphoforms (e.g., phosphorylated serine residues Ser6, Ser9, Ser15, Ser20, Ser37, Ser46, and Ser392). To prevent cell cycle bias relating to p53 staining patterns during DNA replication (21, 44), we conducted our initial experiments using G0-G1-synchronized cells (34).



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  		Figure 1. p53Ser15 nuclear foci form following genotoxic insult from a preexisting p53 pool and form in response to DNA breaks and base damage. A, G0-G1 phase NDF-GM05757 lysates from nonirradiated (NIR) and irradiated (IR; 3 hours after 10 Gy) cultures immunoprecipitated with phospho-specific p53 antibodies followed by Western blotting using a pantropic p53 (Ab-7) antibody. All p53 bands were distinct from the IgG control. B, Western blot analysis of p53Ser15 induction within G0-G1 phase NDF-GM05757 but not in AT-GM05823 fibroblasts. C, representative confocal images of G0-G1 phase NDF-GM05757 cells stained at 3 hours after 10 Gy for pantropic p53 (Ab1801 and DO-1; both red), p53Ser15 (green), and nucleolin (red) and p53Ser15, {gamma}-H2AX, ATMSer1981, DNA-PKcsThr2609, and BRCA1 (all green). All cells were counterstained with DAPI for nuclear DNA (blue). Bar, 5 µm. D, p53Ser15 foci formation in G0-G1 phase NDF-GM05757 fibroblasts at 30 minutes after 10 Gy is similar in the presence of cycloheximide (30 µg/mL) compared with vehicle alone (ethanol)–treated cells. Bar, 20 µm. E, time and dose dependency of protein expression of p53Ser15, p21WAF, {gamma}-H2AX, and actin (loading control) in irradiated G0-G1 phase NDF-GM05757 fibroblasts using Western blot analyses. F, % G0-G1 phase NDF-GM05757 cells positive for p53Ser15 or {gamma}-H2AX foci at 3 hours as a function of radiation dose. G, summary of data in which cells were treated with a variety of DNA-damaging agents and then observed at periods from 30 to 360 minutes following treatment for evidence of p53Ser15 foci. Consistent with p53Ser15 foci being maximally induced by DNA breaks and DNA base damage (i.e., bleomycin, H2O2, hydroxyurea, and MMS; ref. 21) in an ATM-dependent manner (top). +++, dim-to-bright, intermediate-to-large sized foci in >80% of cells; ++, dim-to-bright, intermediate-to-large sized foci in 50% to 80% of cells; +, dim-to-bright, intermediate-to-large sized foci in 10% to 50% of cells; +/–, dim-to-bright, intermediate-to-large sized foci in <10% of cells; –, no p53Ser15 foci, comparable with untreated cells. Representative confocal images of p53Ser15 foci in G0-G1 phase NDF-GM05757 at 3 hours following bleomycin (0.06 unit/mL, 3 hours) and UV (25 J/m2). Bar, 10 µm (bottom).

 
We reasoned that if p53 phosphoforms were involved in DNA damage sensing or recognition, discrete intranuclear foci representing protein aggregates at sites of DNA damage should be observed following irradiation similar to that reported for {gamma}-H2AX (28). Staining with antibodies specific to the Ser15-phosphorylated residue of p53 detected discrete p53Ser15 foci similar in size and morphology to the DNA-dsb-associated foci of {gamma}-H2AX, ATMSer1981, DNA-PKcsThr2609, and BRCA1 (Fig. 1C). In contrast, cell staining with antibodies to pantropic p53 (Ab1801 and DO-1) revealed an accumulation of p53 in a homogenous, nonpunctate nuclear pattern (Fig. 1C), similar to that observed with antibodies directed against p21WAF and pantropic p53 (e.g., Ab421, BP53-12, and Ab-7; data not shown).

Only antibodies recognizing p53Ser15 detected a nonnucleolar and dose-responsive accumulation of p53 foci within 10 minutes following whole-cell irradiation (Fig. 1C, E, and F). Although the other phospho-specific p53 antibodies were able to specifically immunoprecipitate their respective pools of irradiation-activated p53 protein (Fig. 1A), they did not detect increased p53 protein or p53 nuclear foci after irradiation. As such, p53Ser15 was tracked thereafter in subsequent DNA damage experiments. Both p53Ser15 and {gamma}-H2AX expression levels and foci formation were dose-dependent and detected within 10 to 30 minutes after irradiation (Fig. 1E and F; ref. 28), in contrast to the induction of p21WAF, which was maximally induced 3 to 6 hours after irradiation (Fig. 1E). Neither the staining intensity nor induction of p53Ser15 foci was affected by cyclohexamide, an inhibitor of de novo protein synthesis, before irradiation (Fig. 1D). This is consistent with the p53Ser15 signal representing a rapid phosphorylation of a preexisting pool of p53 protein (2).

As ionizing radiation causes several types of DNA lesions within clustered local multiply damaged sites, we used a variety of DNA-damaging agents to determine the lesion specificity for p53Ser15 foci (Fig. 1G). In addition to irradiation, p53Ser15 foci also formed following treatment of G0-G1-synchronized cells with bleomycin, H2O2, and MMS (Fig. 1G). In asynchronously growing NDFs pretreated with hydroxyurea or UV, both agents that can lead to DNA breaks within stalled DNA replication forks, p53Ser15 foci also formed (data not shown; ref. 21). Altogether, these results support that p53Ser15 foci formations are maximally induced by DNA strand breaks. Conversely, in G0-G1-synchronized NDF cells, minimal or no p53Ser15 foci were observed in the first 3 hours following MMC (DNA cross-linking agent) or UV-induced damage (pyrimidine dimers and photoproducts; Fig. 1G). In parallel-treated cultures, cytoplasmic to nuclear translocation and accumulation of total p53 protein was observed following both UV and irradiation (as detected by a pantropic p53 antibody, Ab1801; see Supplementary Fig. S1A). Western blot analyses confirmed delayed and reduced p53Ser15 phosphorylation following UV irradiation of G0-G1-synchronized NDF cells in agreement with the lack of p53Ser15 foci following UV in the absence of stalled replication forks (see Supplementary Fig. S1A). We conclude that the induction of p53Ser15 intranuclear foci is maximally responsive to DNA breaks and DNA base damage within G0-G1 phase cells; a role potentially distinct from that reported for p53 during DNA replication and homologous recombination in S-phase cells (21).

p53Ser15 is a unique chromatin-associated subpool of total p53. To confirm that nuclear p53Ser15 foci are chromatin associated, we treated cells with DNaseI or RNaseA following the preextraction of soluble cellular proteins. Permeabilization removed most of the nucleoplasmic signal of total nuclear p53 protein detected by Ab1801 (Fig. 2A, bottom). In contrast, p53Ser15 was chromatin bound following DNaseI digestion (Fig. 2A, top). The chromatin-associated p53 subpool was similar to the pattern of {gamma}-H2AX staining (Fig. 2A, top). RNaseA treatment also removed only a small proportion of p53Ser15 foci, which may relate to previously described RNA-associated p53 species (14). The NDFs lysates were also biochemically fractionated into cytoplasmic, nuclear, and chromatin-bound fractions (see Materials and Methods). As shown in Fig. 2B, and in agreement with our immunofluorescence data, the p53Ser15 subpool is found within the chromatin-bound fraction similar to the chromatin-bound {gamma}-H2AX. Although total p53 protein can be found in all fractions and increased after irradiation, this is also consistent with our hypothesis that p53Ser15 phosphoforms are a subpool of total p53 protein that is chromatin bound.



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  		Figure 2. Irradiation (IR)–induced p53Ser15 foci are a chromatin-bound subpool of total p53 protein. A, G0-G1 phase NDF-GM05757 cells stained for p53Ser15 at 3 hours after 10Gy with or without preextraction of soluble proteins or treatment with RNaseA or DNaseI. Top, cells are costained for p53Ser15 (green) and {gamma}-H2AX (red). Bottom, total p53 protein (red) detected using Ab1801 antibodies. The extent of each cell nucleus is outlined based on other images of the same cell stained for DAPI (DNA). Bar, 10 µm. B, Western blot analyses of the cytoplasmic, nuclear, and chromatin-bound cellular fractions of asynchronously growing NDF-GM05757 cells before (nonirradiated, NIR) and 2 hours after 10Gy (irradiated, IR) of irradiation. p84 protein was used as a positive control for nuclear protein and {gamma}-H2AX as a positive control for chromatin-bound protein following irradiation. C, immunodepletion analyses of G0-G1 phase NDF-GM05757 lysates before and after irradiation (20 Gy). From these two lysates, total p53 (Total p53) and p53Ser15 (Depleted p53Ser15) were immunoprecipitated with a cocktail of anti-p53 antibodies or p53Ser15 antibody, respectively. Following p53Ser15 immunoprecipitation, the supernatant was subjected to a subsequent immunoprecipitation with the p53 antibody cocktail to determine the amount of remaining total p53 (Remaining p53) after p53Ser15 depletion. Immunoprecipitated protein was detected by Western blot using pantropic p53 (Ab-7).

 
Despite differential staining patterns within the first 3 hours after 10 Gy of irradiation (Fig. 1C), at 24 hours, we observed some foci formation by pantropic p53 antibodies (as detected by either Ab1801 or FL393; see Supplementary Fig. S2A). This Ab1801 subpopulation significantly colocalized with residual p53Ser15 foci (see Supplementary Figs. S1B and S2), consistent with Ab1801 detecting residual p53Ser15 foci and with p53Ser15 being a subpool of total p53. Further analyses by immunoprecipitation and immunodepletion of cell extracts confirmed this finding (Fig. 2C). The relative levels of total p53, p53Ser15, and remaining p53 following p53Ser15 depletion were determined and were consistent with a ratio of 0.25 for p53Ser15 protein to total p53 protein. This ratio increased to 0.5 at 24 hours after irradiation (Fig. 2C). Taken together, these results support p53Ser15 protein as a distinct chromatin-associated subpool of total p53 during the DNA damage response.

Dependency and association of p53Ser15 foci on ataxia-telangiectasia mutated, DNA-dependent protein kinase catalytic subunit, or MRE11/RAD50/NBS1 function. Initial microscopic control experiments were undertaken as a means to document the formation of p53Ser15 as a function of upstream kinases. As predicted, p53Ser15 foci formation was attenuated or delayed in irradiated AT-GM05823, AT-GM02052 fibroblasts, and NDF-GM05757 fibroblasts pretreated with wortmannin at concentrations that inhibit the ATM kinase activity (Fig. 1B, Fig. 3A and B). p53Ser15 foci formation was observed in the DNA-PKcs-deficient MO59J glioblastoma cell line, consistent with a primary role for ATM as the PI3K responsible for the phosphorylation of p53Ser15 (data not shown; refs. 24, 28). We observed delayed p53Ser15 foci formation in the NBS-GM07166 fibroblast strain (Fig. 3A and B, right) most probably reflecting the need for intact MRN signaling upstream of ATM-mediated phosphorylation (5, 32). Both p53Ser15 protein levels and foci were elevated before and after irradiation in the NHEJ-defective 180BR fibroblast strain, consistent with residual DNA breaks being associated with residual p53Ser15 foci at 24 hours following DNA damage. In addition, we also observed colocalized p53Ser15 and {gamma}-H2AX foci within acentric chromosome fragments contained within micronuclei in irradiated 180BR cells. This observation is consistent with residual foci being a manifestation of nonrepaired breaks (Fig. 3A and B, right; ref. 45).




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  		Figure 3. p53Ser15 foci dependency and association with ATM, DNA-PKcs, {gamma}-H2AX, and the MRN complex. A, representative confocal images of p53Ser15 (green) and {gamma}-H2AX (red) foci formation in G0-G1 phase NDF-GM05757, NBS-GM07166, and 180BR (DNA Ligase IV deficient) fibroblasts at 3 hours following 10 Gy. Inset, arrows, micronuclei containing colocalized p53Ser15 and {gamma}-H2AX (yellow). No p53Ser15 foci are observed following pretreatment of NDF-GM05757 cells with the PI3K inhibitor wortmannin or in irradiated AT-GM05823 fibroblasts. For NDF-GM05757 strains, nuclei are also counterstained with DAPI to delineate nuclear DNA. Bar, 20 µm. B, % G0-G1 phase NDF-GM05757, AT-GM05823, NBS-GM07166, and 180BR cells with more than two p53Ser15 foci over time after irradiation showing a dose-response in the NDF cells. Right, deficient or delayed foci formation in AT or NBS cells and increased endogenous and late residual foci formation in DNA Ligase IV–deficient 180BR cells. C, representative confocal images of G0-G1 phase NDF-GM05757 nuclei costained with p53Ser15, {gamma}-H2AX, and 53BP1at 3 hours following 10 Gy. D, costaining of p53Ser15, RAD50, and MRE11 was conducted at 6 hours following 10 Gy. Colocalization between foci in merged images appears as yellow foci. Dotted line outlines the cells' nuclei based on DAPI-DNA staining. Bar, 5µm. Colocalization was confirmed with (a) fluorescent intensity profiles (see coincident red-green intensity profiles within nuclear regions traversed by white line; middle) and (b) separate calculations of Pearson correlation coefficients for colocalization (rp) based on the whole nucleus (bottom). Control data for the colocalization of the MRN complex (Supplementary Fig. S3). E, G0-G1 phase NDF-GM05757 nuclei costained with either {gamma}-H2AX and DNA-PKcsThr2609 (1 hour following 1 Gy) or {gamma}-H2AX and p53Ser15 (3 hours following 2 Gy). Left three, single confocal sections. Right single, three-dimensional rendered reconstructed from Z stacks of confocal sections. Yellow voxels, volumes within foci that are colocalized. Bar, 2 µm. F, % 53BP1 foci colocalized with {gamma}-H2AX and p53Ser15 foci in G0-G1 phase NDF-GM05757 nuclei at 3 hours following 10 Gy. G, % cells with more than two colocalized p53Ser15/RAD50 foci as a function of time and dose in G0-G1 phase NDF-GM05757 nuclei. H, confirmation of biochemical interaction between p53Ser15 and {gamma}-H2AX using coimmunoprecipitation/Western blot analyses in G0-G1 phase NDF-GM05757 or asynchronous HCT116-p53+/+ or HCT116-p53-/- cells (left and middle). All bands detected were specific and distinct from the IgG and beads-only controls. Western blot of p53Ser15 and {gamma}-H2AX expression in HCT116 p53+/+ and HCT116 p53-/- cell lysates (right).

 
We next determined whether p53Ser15 foci would colocalize with {gamma}-H2AX/53BP1, MRN, and PML-associated complexes following whole-cell irradiation (2, 28, 46). Although 53BP1 has been shown to bind p53 in yeast two-hybrid and crystallization studies (8), there are few data reporting p53 and 53BP1 colocalization in non–S-phase cells in vivo (21). Using microscopic and statistical analyses, we observed a significant induction of colocalization of ~30% to 60% of p53Ser15 foci with {gamma}-H2AX, 53BP1, RAD50, and MRE11 foci following whole-cell irradiation, in a time-dependent and dose-dependent manner (Fig. 3C-G). For comparison, in the same experiment, there was almost a 1:1 colocalization between 53BP1 and {gamma}-H2AX at 3 hours after 10 Gy (Fig. 3C and E), and colocalization between RAD50 and NBS1 was observed at 6 hours after 10 Gy (data not shown; Supplementary Fig. 3B). Colocalized foci were specifically chosen to highlight the coincident peaks of the respective fluorescent signals based on the line scan plots beneath the corresponding images in Fig. 3C and D. In separate analyses based on focal staining patterns within the entire nucleus, Pearson correlation coefficient calculations (see Materials and Methods, as described by Rubbi and Milner; ref. 39) confirmed significant, nonrandom colocalizations between p53Ser15 foci and {gamma}-H2AX, 53BP1, RAD50, and MRE11 foci. In addition, Z stacks of the intranuclear confocal sections were also collected and used to reconstruct the nuclei with foci in three dimensional space, which also confirmed that the foci were colocalized in all three nuclear planes (Fig. 3E). Although there can be almost a 1:1 colocalization observed between {gamma}-H2AX and 53BP1 and {gamma}-H2AX and DNA-PKcsThr2609 (47), the colocalization of p53Ser15 and any of {gamma}-H2AX, 53BP1, RAD50, and MRE11 is less than this. This may reflect differences in the kinetics of p53Ser15 binding over time (e.g., transient interactions) with chromatin-associated sites of DNA damage. In our study, p53Ser15 and PML foci colocalized minimally (Supplementary Fig. 3E), suggesting that these p53Ser15 foci are a separate subpool to that described as interacting with PML during cellular senescence (48).

We were able to biochemically confirm a direct p53Ser15/{gamma}-H2AX interaction by coimmunoprecipitating endogenous p53Ser15 and {gamma}-H2AX in vivo from both G0-G1-synchronized NDF-GM05757 fibroblasts and asynchronously growing HCT116 p53+/+ colorectal cancer cells following DNA damage (Fig. 3H). Maximal amounts of p53Ser15 and {gamma}-H2AX were coimmunoprecipitated at 3 hours and then decreased at 24 hours after irradiation (Fig. 3H); these data are consistent with observed kinetics of p53Ser15 foci formation and resolution.

p53Ser15 kinetics correlate with biochemical DNA double-strand break rejoining. Quantitative microscopy confirmed that p53Ser15 and {gamma}-H2AX foci had similar kinetics of induction and resolution over the first and subsequent hours following irradiation (Fig. 4A and B). These data are consistent with the Western blot analyses in Fig. 1E. The residual number of {gamma}-H2AX foci at 24 hours was similar to that of the residual number of p53Ser15 foci after 2 and 10 Gy (Fig. 4A and B). To ascertain relative foci formation following both low (2 Gy) and high (10 Gy) doses, we used confocal microscopy. Typical nuclear depths for G0-G1 NDF-GM05757 cells are ~5 µm, which is equivalent to two to three 1.8-µm confocal sections through the nucleus. Therefore, the total number of p53Ser15 and {gamma}-H2AX residual foci at 24 hours following 2 or 10 Gy is estimated at 3 to 5 or 15 to 25 foci per nucleus. This approximates the predicted 2.5 to 5 DNA-dsbs per Gy at 24 hours following irradiation (based on 5-10% residual DNA breaks remaining at 24 hours as observed by continuous field gel electrophoresis; see Fig. 4C).



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  		Figure 4. p53Ser15 nuclear foci correlate with DNA-dsb rejoining and are resistant to proteolytic degradation. A, mean number of {gamma}-H2AX foci per 1.8-µm confocal section in G0-G1 phase NDF-GM05757 cells as a function of time and dose (2 or 10 Gy). B, mean number of p53Ser15 foci per 1.8-µm confocal section in G0-G1 phase NDF-GM05757 cells as a function of time and dose (2 or 10 Gy). C, biochemical DNA-dsb rejoining kinetics in G0-G1 phase NDF-GM05757 fibroblasts using CFGE, plotted as the fraction of DNA-dsbs remaining (FDR) versus time. Note the ~5% to 10% of initial DNA-dsbs remaining at 24 hours after irradiation. D, quantitation of the mean number of p53Ser15 nuclear foci in G0-G1 phase NDF-GM05757 cells over 24 hours in the presence of the 26S proteasome inhibitor, MG132, compared with vehicle-alone control (DMSO)–treated cells.

 
In the absence of DNA damage, the short half-life of endogenous p53 protein is regulated by the MDM2 protein, an E3 ubiquitin ligase, which targets p53 for 26S-proteosomal degradation (49). If p53Ser15 foci were associated with DNA damage sensing or repair, then the observed kinetics of this irradiation-phosphorylated nuclear p53 pool should be resistant to proteasomal-mediated degradation and also distinct from nucleolar p53 subpools (Fig. 1C; ref. 39). We therefore investigated whether p53Ser15 foci formation is altered in NDF cells treated with MG132, an inhibitor of the 26S proteosome. Consistent with previous data (50), neither DMSO nor MG132 induced p53Ser15 in the absence of DNA damage. We observed in both the DMSO control– and MG132-treated cells an increase at 3 hours followed by a decrease in p53Ser15 foci and protein levels over 24 hours (Fig. 4D). We conclude that p53Ser15 foci induction and resolution represents a dose-responsive nuclear p53 subpool that is resistant to proteasomal degradation.

Lack of Ser15-phosphorylation leads to altered DNA rejoining, foci formation, and {gamma}-H2AX association following irradiation-induced DNA damage. To determine whether Ser15 phosphorylation and G1 checkpoint control could functionally affect DNA-dsb sensing and/or repair, we used MEFs that expressed a knock-in p53 mutation at murine Ser18 (p53Ser18Ala/Ser18Ala), whereby the mouse Ser18 residue is homologous and functionally equivalent to human Ser15 residue. This knock-in phosphorylation-deficient mutation does not affect p53 stabilization or DNA binding (35). Using the COMET assay to afford single-cell analysis, we observed increased residual DNA-dsbs following doses in excess of 20 Gy in the p53Ser18Ala/Ser18Ala MEFs compared with p53WT/WT MEFs (Fig. 5A), suggesting that local multiply damaged sites following irradiation may be less effectively sensed in the mutant cells. High levels of nonspecific p53-staining in MEF cells precluded correlative microscopy of p53Ser15 and {gamma}-H2AX in these experiments.



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  		Figure 5. Lack of Ser15 phosphorylation leads to altered DNA-dsb rejoining, foci formation, and {gamma}-H2AX association following irradiation (IR)–induced DNA damage. A, biochemical DNA-dsb rejoining in asynchronous isogenic p53WT/WT or p53Ser18Ala/ Ser18Ala MEFs as determined from the COMET assay expressed as the fraction of DNA-dsbs released (FDR) over time following 20 Gy. B, representative confocal images of HCT116-p53–/– cells transiently transfected with YFP alone (control), YFP-p53WT and YFP-p53Ser15Ala fusion constructs before and after irradiation (3 hours following 5 Gy). Bar, 5 µm. C, Western blot analyses of lysates before and after irradiation (3 hours following 5 Gy) from HCT116-p53+/+ cells transiently transfected with YFP-p53WT and YFP-p53Ser15Ala. D, coimmunoprecipitation analyses using YFP and {gamma}-H2AX antibodies to detect YFP-p53 fusion proteins in complex with {gamma}-H2AX before and after 5 Gy (3 hours). Negative controls of YFP-vector expressing cells and IgG alone immunoprecipitations showed no interaction with {gamma}-H2AX (data not shown).

 
To determine whether foci formation is directly affected by p53Ser15 phosphorylation, transient transfection studies of HCT116 p53–/– human carcinoma cells with human YFP-p53 phosphomutant constructs were done using DNA sequences encoding wild-type p53 or p53 mutated from serine to alanine at residue 15. Control YFP alone and YFP-p53Ser15Ala protein expression did not lead to foci formation in the absence or presence of DNA damage induction. This is in contrast to YFP-p53WT expression in which foci are induced following DNA damage (Fig. 5B). Due to toxicity concerns using p53WT constructs in null-p53 cells, we also used HCT116 p53+/+ human carcinoma cells to transiently transfect p53 fusion constructs and determine their association with {gamma}-H2AX following DNA damage. YFP-p53WT levels were stabilized at 3 hours after 10 Gy with an associated induction of p21WAF and {gamma}-H2AX (Fig. 5C). In contrast, YFP-p53Ser15Ala phosphomutant levels remained unchanged and resulted in relatively reduced levels of p21WAF protein levels following irradiation. {gamma}-H2AX induction was similar in both YFP-p53WT- and p53Ser15Ala-expressing cells (Fig. 5C). Finally, in agreement with our endogenous data using NDF cells, we observed an increased biochemical association of {gamma}-H2AX and YFP-p53WT at 3 hours after irradiation but not for YFP-p53Ser15Ala (Fig. 5D). Taken together, our data is consistent with a role for p53 Ser15 phosphorylation as a mediator of chromatin binding to {gamma}-H2AX megabase domains during DNA-dsb sensing and repair.

Interaction between p53Ser15, ATMSer1981, and DNA-PKcsThr2609 at focal DNA damage. Given the morphologic similarity between p53Ser15 foci and that of {gamma}-H2AX, ATMSer1981, and DNA-PKcsThr2609 foci, we next determined whether p53Ser15 could directly colocalize at discrete sites of DNA-dsbs in vivo within the first hour during DNA-dsb signaling. We initially used a high-LET helium-3 ion microbeam to irradiate discrete subnuclear areas of <5-µm diameter with minimal scatter (detailed by Belyakov et al.; ref. 37). Using this microbeam technique, between 4 and 6 DNA-dsbs per helium ion are created within the dense ionization cluster of the irradiated cylindrical nuclear volume. Within targeted G0-G1 NDF-GM05757 cells, at 30 minutes following a dose of 1 to 100 helium-3 ions, a discrete three-dimensional {gamma}-H2AX focus was observed and colocalized with 53BP1 (Fig. 6A). In similar-targeted cells, p53Ser15 and ATM were also observed to colocalize within the discrete irradiated cylindrical volume of nuclear damage (Fig. 6A).



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  		Figure 6. Interaction in vivo between p53Ser15, {gamma}-H2AX, ATMSer1981, and DNA-PKcsThr2609 at DNA breaks. A, G0-G1 phase NDF-GM05757 nuclei after 30 minutes of recovery following subnuclear microbeam irradiation with 100-targeted helium ions. Confocal images in both X-Y and Y-Z planes confirmed focal colocalizations within targeted nuclear volumes between {gamma}-H2AX/53BP1 and p53Ser15/ATM. Bar, 5 µm. (B-D) Wide-field immunofluorescent microscope images of asynchronous NDF-GM05757 nuclei at various times following UV laser microbeam irradiation showing the colocalization between (B) {gamma}-H2AX, DNA-PKcsThr2609, and ATMSer1981 at sites of DNA-dsbs, as detected by TUNEL; (C) DNA-PKcsThr2609 with ATMSer1981 and p53Ser15 with DNA-PKcsThr2609; and (D) p53Ser15 with {gamma}-H2AX and ATMSer1981. Arrows, traversal of the UV laser beam. In similar experiments, both ATMSer1981 and p53Ser15 staining was absent in AT-GM05823 (data not shown). Bar, 10 µm. E, proposed model for p53Ser15 in irradiation (IR)–induced DNA damage sensing. Within minutes following cellular irradiation, DNA damage sensing and repair proteins are recruited to sites of DNA damage. Both ATM and DNA-PKcs undergo autophosphorylation in response to DNA breaks leading to phosphorylation of H2AX. Upon binding to damaged chromatin, ATM directly phosphorylates p53Ser15, 53BP1, and CHK2Thr68 close to or within chromatin sites containing {gamma}-H2AX for varying time intervals. Subsequent p53-mediated transactivation can occur in a pan-nuclear fashion at a distance from damaged DNA. Phosphorylation of Ser15 on p53 also blocks MDM2-mediated p53 degradation and nuclear export allowing for increased nuclear p53Ser15 concentration to facilitate optimal DNA damage signal transduction within the nucleus. Cells that are defective in DNA-dsb rejoining or have residual DNA-dsb 24 hours following irradiation show residual p53Ser15 foci as an indicator of persistent DNA damage and genetic instability.

 
A similar discrete recruitment of p53Ser15 was observed within subnuclear tracks of DNA-dsbs created with a UV laser microbeam technique (Fig. 6C and D; ref. 28). Within 10 minutes of recovery, we consistently observed {gamma}-H2AX signal accumulating along terminal deoxynucleotidyl transferase–mediated nick-end labeling (TUNEL)–positive tracks (Fig. 6B). DNA-PKcsThr2609 and ATMSer1981 also colocalized within 10 minutes with {gamma}-H2AX and TUNEL tracks (Fig. 6B), supporting recent data in which ATM and DNA-PKcs were shown to redundantly phosphorylate {gamma}-H2AX and p53Ser15 and that ATM is recruited quickly to DNA-dsbs (1, 2, 29). Subsequently, using the {gamma}-H2AX and DNA-PKcsThr2609 signals as indicators of UV laser–induced DNA-dsbs, we observed p53Ser15 accumulation within these tracks as early as 10 minutes following irradiation (Fig. 6C) with maximal recruitment of p53Ser15 occurring at 30 to 60 minutes after irradiation (Fig. 6D). We conclude that p53Ser15 binds directly to damaged chromatin domains containing {gamma}-H2AX, 53BP1, ATMSer1981, and DNA-PKcsThr2609 in the first hour during the sensing and repair of DNA-dsbs.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The p53 tumor suppressor protein achieves a broad realm of cellular functions through a series of discrete and well-timed post-translational modifications to protect against cellular carcinogenesis. These initiate its well-characterized function as a transcriptional transactivator and a mediator of cell cycle checkpoint control and cell death following DNA damage (13). Our study is unique in that it documents temporal data regarding specific p53 phosphoforms in response to DNA damage in vivo under physiologic conditions in primary human fibroblasts. The data herein indicates that p53Ser15 phosphoforms are a unique subpool of total cellular p53 (39) and are chromatin-associated at sites of DNA breaks and {gamma}-H2AX megabase domains. However, this p53Ser15 subpool most probably possesses other post-translational modifications given the interdependency of phosphorylation and acetylation (11), and these will require further study.

Our kinetic, colocalization and coimmunoprecipitation data indicate that the preexisting "latent" subpool of nuclear p53 is rapidly phosphorylated in vivo at {gamma}-H2AX domains and is dependent on ATM and MRN but not DNA-PKcs. The recruitment of latent p53 that is phosphorylated within minutes following DNA damage is consistent with the high affinity of latent p53 to bind DNA in a nonspecific manner, which may relate to additional chromatin binding at DNA breaks rather than solely at p53 consensus sequences within downstream target genes (Fig. 2A; ref. 51). Nonetheless, in response to genotoxic stress, p53 has been recently localized at transcription sites, including that of p21WAF, and is phosphorylated by hSMG-1 during RNA processing (4, 13, 14). Our observation that there are qualitatively less p53Ser15 foci following RNaseA treatment (Fig. 2A) could be consistent with a proportion of p53Ser15 at sites of transcription or associated with RNA processing. Further experiments are required to clarify the relative extent and dynamics of chromatin-bound versus RNA-bound p53 phosphoforms following genotoxic insult. The delayed kinetics of p53Ser15 foci formation when compared with {gamma}-H2AX foci formation reflects the requirement of initial ATM phosphorylation but may also reflect a secondary recruitment of p53 to clustered damage within local multiple damaged sites or DNA breaks created during DNA-dsb, DNA-ssb, or base excision repair lesion processing (25). We speculate that the number, site, and nature of p53Ser15 interactions may serve as a counting mechanism for cells to assess the quantity, type, and severity of damage to mediate cell cycle checkpoint control in preventing cellular carcinogenesis.

We observed less than 1:1 colocalization of p53Ser15 with {gamma}-H2AX in contrast to other chromatin-associated proteins, such as 53BP1 (Fig. 3C and E). This may reflect a transient p53Ser15/{gamma}-H2AX interaction, similar to that reported for CHK2. In this case, initial direct recognition of DNA damage by MRN and ATM is subsequently followed by phosphorylation of p53 and CHK2 at damaged chromatin before initiation of downstream signaling events throughout the nucleus (18, 19). Based on our data, we propose a transient interaction model in which p53Ser15 initially localizes at sites of {gamma}-H2AX-associated megabase domains (Fig. 6E); this interaction could be mediated by the p53 nonspecific DNA binding domain in the COOH terminus and involve linear protein-chromatin diffusion (Fig. 6E; refs. 21, 51, 52). Although the requirement of NH2-terminal phosphoserine residues for all of the transcriptional trans-activation by p53 remains controversial, phosphorylation of the Ser15 and other residues may be required for nuclear retention and/or immediate binding to damaged chromatin in addition to downstream gene activation (e.g., p21WAF; refs. 21, 51, 52). As DNA repair ensues, chromatin-bound "activated" p53Ser15 phosphoform may be dephosphorylated similar to the dephosphorylation of {gamma}-H2AX and DNA-PKcs by PP1 and PP5, respectively (53), or be released into the nucleoplasm to transactivate downstream p53 target genes at a distance from the original binding site of DNA damage (14, 18).

The observed ATM-p53 interactions at DNA breaks supports previous chromatin immunoprecipitations that determined murine ATM and p53Ser18 form a complex at sites of DNA-dsbs during V(D)J recombination and microscopy studies in which ATM can colocalize to {gamma}-H2AX domains (26, 29, 54). Our observation of colocalized ATMSer1981 and DNA-PKcsThr2609 at sites of DNA breaks is also corroborated by the recent finding that both kinases redundantly phosphorylate {gamma}-H2AX (1). Our finding that p53Ser15 and DNA-PKcsThr2609 colocalize at sites of DNA breaks in situ in G1 cells is consistent with the concept that phosphorylated p53 species may act as a mediator between checkpoint control and NHEJ during the G1 phase of the cell cycle. This is also consistent with DNA-PKcs and CHK2 synergistically activating preexisting p53 following DNA damage (3, 9). Additionally, the importance of the Ser15 residue in our YFP-p53 transfection assays is strengthened by previous data in which NHEJ is stimulated in vitro by the addition of recombinant p53Asp15 (which mimics a constitutively phosphorylated p53Ser15) but not by recombinant phosphorylation-inhibited p53Ala15 (55). Taken together with reports of a role for MDM2 in DNA-dsb repair, p53Ser15 complexed with BLM helicase at DNA replication forks, and the observation of p21WAF foci at sites of subnuclear damage (21, 56, 57), these localized chromatin-bound proteins associated with the ATM-p53 signaling axis may interact at an exquisite local level near DNA-damaged sites. These interactions may amplify subsequent intracellular signaling secondary to the DNA breaks to initiate the G1 checkpoint in protecting genomic stability.

The fact that unlike ATM–/– MEFs, the p53Ser18Ala/Ser18Ala MEFs (35) are not radiosensitive, allows us to conclude that Ser15 phosphorylation and/or a deficient G1 checkpoint are not major factors of cell survival following DNA damage (21). Indeed, the consequences of DNA damage sensing by p53Ser15 may relate more to the fidelity rather than the overall level of DNA repair as a mediator of genetic stability. This role was recently supported by the finding that wild-type p53 reduces error-prone NHEJ DNA-dsb repair (based on interchromosomal reporter substrates) in MEF cells and may protect against carcinogenesis (22, 35). Cells defective in Ser392 and Ser389 phosphorylation of p53, in combination either with p53Arg175His or p53Arg248Trp hotspot mutations, acquire increased cellular transformation in vitro (16, 58, 59). In a similar manner, abrogated Ser15 phosphorylation, observed in many mutant p53 proteins (20), may also affect mutagen-induced rates of transformation in combination with p53 hotspot mutations; a hypothesis currently under study in our laboratory. The fact that ATM-dependent p53 localization to centrosomes during the post-mitotic checkpoint also requires Ser15 phosphorylation attests to a broad role for p53 modifications in preventing aneuploidy and protecting against genetic instability through multiple mechanisms (60). Discordance between DNA-dsb sensing/repair and cell cycle checkpoint control, in the absence of cell death signals, may be one factor in the selection of mutant clones during the process of cellular carcinogenesis. Our studies may partially explain the relative negative prognosis for mutant p53-expressing tumors, which would exhibit defects in p53Ser15 signaling and DNA-specific binding and lead to the selection of clones that exhibit therapeutic resistance (20).


   Acknowledgments
 
Grant support: Cancer Research UK (K.M. Prise), Gray Cancer Institute (K.M. Prise), Canadian Institutes of Health Research (D.P. Bazett-Jones), U.S. Department of Energy grants C50519 and CA86936 (D.J. Chen), National Cancer Institute of Canada grant 14246 (R.G. Bristow), Canadian Foundation of Innovation (R.G. Bristow), PMH Foundation (R.G. Bristow), and NCIC Ph.D. Research Studentship award (S.T. Al Rashid).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank S. Benchimol, C. Arrowsmith, S. Powell, P. Olive, L. Harrington, J. Danska, B. Marples, R. Kanaar, and P. Bradshaw for protocols and critical comments and S. Gilchrist, B. Sutherland, J. Jonkman, and S. Rahman for technical assistance.


   Footnotes
 
Note: G. Dellaire is a Senior Postdoctoral Fellow of the Canadian Institutes of Health Research. R.G. Bristow is a Canadian Cancer Society Research Scientist.

Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 3/ 3/05. Revised 7/ 7/05. Accepted 9/12/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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