Histone H2AX Is Phosphorylated in an ATR-dependent Manner in Response to Replicational Stress

Irene M. Ward, Junjie Chen
2001 Journal of Biological Chemistry  
H2AX, a member of the histone H2A family, is rapidly phosphorylated in response to ionizing radiation. This phosphorylation, at an evolutionary conserved C-terminal phosphatidylinositol 3-OH-kinase-related kinase (PI3KK) motif, is thought to be critical for recognition and repair of DNA double strand breaks. Here we report that inhibition of DNA replication by hydroxyurea or ultraviolet irradiation also induces phosphorylation and foci formation of H2AX. These phospho-H2AX foci colocalize with
more » ... ci colocalize with proliferating cell nuclear antigen (PCNA), BRCA1, and 53BP1 at the arrested replication fork in S phase cells. This response is ATR-dependent but does not require ATM or Hus1. Our findings suggest that, in addition to its role in the recognition and repair of double strand breaks, H2AX also participates in the surveillance of DNA replication. Histone H2AX has been implicated in the maintenance of genomic stability in response to DNA double strand breaks (DSBs). 1 It is phosphorylated at an evolutionary conserved phosphatidylinositol 3-OH-kinase-related kinase (PI3KK) motif in the carboxyl terminus within seconds after exposure to ionizing radiation (IR) (1). Immunofluorescence studies have revealed that phosphorylated H2AX (␥-H2AX) forms nuclear foci at the sites of DSBs. These foci appear within 1 min after exposure of cells to IR. Their numbers increase in the first 10 -30 min after irradiation before they gradually decline correlating with the predicted value of slowly re-joining DSBs (2). ␥-H2AX foci are also found at sites of V(D)J recombinationinduced DSBs in developing thymocytes (3) and at sites of recombinational DSBs during meiosis (4). In addition, phosphorylation of H2AX is also induced by initiation of DNA fragmentation during apoptosis (5). Thus, H2AX is phosphorylated in response to DSBs. Several proteins involved in the DNA damage response (e.g. 53BP1, Rad50, NBS1, MRE11, BRCA1) have been shown to subsequently colocalize with ␥-H2AX at the sites of strand breaks in response to IR (6 -8). Prevention of H2AX phosphorylation by treatment with the PI3KK inhibitor wortmannin eliminated the foci formation of other repair proteins suggesting a role of ␥-H2AX in the recruitment of repair factors to sites of DNA double strand breaks (6). Furthermore, mutations in the C-terminal PI3KK motif of Saccharomyces cerevisiae histone H2A1 resulted in hypersensitivity to agents that lead to DNA double strand breaks (9). Taken together, these results strongly suggest that H2AX is required for the recognition and repair of DNA double strand breaks. The roles of H2AX in the cellular responses to replication block and/or other types of DNA damage have not yet been studied. Surveillance of DNA replication and progression through the cell cycle is regulated by checkpoints that ensure the temporal coordination of critical cell cycle events. The S phase checkpoint prevents the initiation of mitosis until DNA replication is completed. This checkpoint was originally described in the budding yeast S. cerevisiae by the isolation of loss of function mutants that initiate mitosis despite an HU-induced replication block (10). One of the proteins that control HU-mediated replication arrest in S. cerevisiae is the PI3Krelated kinase MEC1, the homologue of mammalian ATR. ATR null mice die early in embryogenesis and ATRϪ/Ϫ blastocytes show a phenotype resembling mitotic catastrophe (11, 12). Recently, it has been shown that ATR forms foci at the sites of stalled replication forks in response to replication arrest (13). These foci overlap with foci formed by the product of the breast cancer susceptibility gene BRCA1 (13). Furthermore, BRCA1 is phosphorylated by ATR following exposure to HU or UV (13, 14) . Taken together, these data suggest that ATR and BRCA1 are involved in the DNA replication checkpoint control. Here we show that replicational stress due to HU or UV treatment also induces phosphorylation and foci formation of H2AX. These ␥-H2AX foci colocalize with proliferating cell nuclear antigen (PCNA) and BRCA1 at the arrested replication fork in S phase cells. Cells lacking wild-type ATM (ataxiatelangiectasia-mutated) showed no difference in HU-induced ␥-H2AX foci formation nor was the response impaired in Hus1 (hydroxyurea sensitive)-deficient cells. In contrast, overexpession of kinase-inactive ATR (ataxia-telangiectasia-Rad3-related) inhibited the phosphorylation and foci formation of H2AX upon treatment with HU or UV, suggesting that H2AX is phosphorylated by ATR in response to replication blocks. EXPERIMENTAL PROCEDURES Cell Culture and Genotoxic Agents-Human cell lines were grown in RPMI 1640 supplemented with 10% fetal bovine serum (FBS). Hus1Ϫ/ Ϫp21/or Hus1ϩp21Ϫ/Ϫ mouse fibroblasts were grown in Dulbecco's modified Eagle's medium plus 10% FBS. For cell cycle experiments, MCF-7 cells were synchronized by serum starvation for 24 h and release into RPMI plus 20% serum (15). Irradiation was performed using a 137 Cs source, UV light was delivered in a single pulse using a Stratalinker, and HU was added to final volume of 2 mM. If not indicated otherwise, the cells were harvested 1 h after drug application or exposure to IR and UV. Antibodies-Rabbit polyclonal anti-␥-H2AX, mouse monoclonal anti-BRCA1, and mouse monoclonal anti-53BP1 antibodies were raised as described previously (7) . The anti-PCNA specific antibody was purchased from Santa Cruz Biotechnology, Inc. Immunostaining-Cells grown on coverslips were fixed for 5 min in a 1:1 methanol:acetone solution prior incubation with the primary antibodies for 20 min at 37°C. Fluorescein isothiocyanate-conjugated goat . 1 The abbreviations used are: DSB(s), double strand break(s); PI3K, phosphatidylinositol 3-kinase; PI3KK, phosphatidylinositol 3-OH-kinase-related kinase; IR, ionizing radiation; HU, hydroxyurea; PCNA, proliferating cell nuclear antigen; FBS, fetal bovine serum. This paper is available on line at http://www.jbc.org 47759 by guest on July 24, 2018 http://www.jbc.org/ Downloaded from anti-mouse and/or rhodamine-conjugated goat anti-rabbit serum (Jackson ImmunoResearch) were used as secondary antibodies. All antibodies were diluted in 5% goat serum. Cells were counterstained with Hoechst dye for 30 s, mounted, and viewed with a Nikon ECLIPSE E800 fluorescence microscope using a 60ϫ objective. Images were processed using Adobe Photoshop and Canvas software. Western Blotting-Cells were lysed in NETN buffer (150 mM NaCl, 1 mM EDTA, 20 mM Tris (pH 8), 0.5% Nonidet P-40), and the insoluble fraction was pelleted for 5 min in a microcentrifuge. Histones were extracted from the pellets with 0.1 M HCl, boiled in SDS gel sample buffer, and loaded on a 15% or 17% SDS-polyacrylamide gel. The separated proteins were transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore). The membrane was blocked with 5% nonfat milk for 30-min prior incubation with 50 ng/ml anti-␥-H2AX antibodies for 1 h. The blots were washed in TBST (10 mM Tris-Cl (pH 8), 150 mM NaCl, 0.2% Tween 20), incubated with horseradish peroxidaseconjugated protein A, (Amersham Pharmacia Biotech) and visualized by chemiluminescence using the Supersignal kit purchased from Pierce. RESULTS H2AX Is Phosphorylated at the Sites of Stalled Replication in Response to HU and UV Treatment-To investigate the phosphorylation and foci formation of H2AX we raised polyclonal anti-phospho-H2AX antibodies against a C-terminal peptide containing phosphorylated Ser-139. Results obtained by immunofluorescence studies and Western blotting demonstrated that these antibodies specifically recognize phosphorylated H2AX and do not cross-react with the unphosphorylated form (7). As described earlier for various cell lines (2, 7, 8), exposure of HBL100 cells to IR resulted in the rapid and dose-dependent formation of ␥-H2AX foci ( Fig. 1A and data not shown) . In addition, extensive ␥-H2AX staining was observed in a subpopulation of cells treated with 2 mM hydroxyurea or low dose (10 J/m 2 ) UV radiation (Fig. 1A) . Western blotting analyses confirmed that H2AX is phosphorylated in response to HU or UV treatment (Fig. 1B) . HU blocks DNA replication by inhibiting ribonucleotide reductase, which results in decreased intracellular deoxynucleoside triphosphates that are required for DNA synthesis. Similarly, exposure of S phase cells to UV results in replication arrest, since most DNA polymerases are unable to replicate templates containing UV-induced DNA lesions (16). We therefore asked whether the observed ␥-H2AX staining is restricted to the arrested DNA replication forks. Coimmunostaining with PCNA revealed that H2AX is phosphorylated at the replication fork upon treatment with HU (Fig. 1C) . A similar response was observed after exposure to 10 J/m 2 UV (Fig. 1C) , while a high dose of UV radiation (100 J/m 2 ), which introduces multiple types of DNA damage, induced ␥-H2AX foci formation both in S phase and non-S phase cells (data not shown). BRCA1 has been reported to form nuclear foci at the sites of stalled replication forks (15). Coimmunostaining analyses revealed that ␥-H2AX foci greatly overlap with BRCA1 foci in response to HU treatment (Fig. 1D) . We have demonstrated previously that p53 binding protein-1 (53BP1) also forms nuclear foci in response to replication arrest (7). As shown in Fig. 1D , lower panel, these 53BP1 foci colocalize with ␥-H2AX following treatment with HU. Thus, ␥-H2AX colocalizes with PCNA, BRCA1, and 53BP1 at the arrested replication forks. To further confirm that the phosphorylation of H2AX upon exposure to HU or low dose UV is related to DNA replication, we synchronized MCF7 cells by a cycle of serum starvation and release into high serum. Only a few cells showed ␥-H2AX staining when exposed to HU or UV 12 h after release into high serum when the majority of cells are found in the G 1 phase of the cell cycle (Fig. 2, left panel) . In contrast, most cells showed HU-or UV-induced phosphorylation of H2AX when analyzed during early or late S phase (24 and 36 h after serum release, respectively) correlating with the pattern of PCNA staining (Fig. 2) . While phosphorylation of H2AX in response to HU or UV treatment proofed to be cell cycle-dependent, exposure to IR induced H2AX phosphorylation and foci formation both in S phase and G 1 phase cells (Fig. 2 ). Together these results suggest that H2AX is phosphorylated at the replication fork in response to replication block. H2AX Phosphorylation in Response to Replication Arrest Requires ATR but Is Independent of ATM and Hus1-Although FIG.
doi:10.1074/jbc.c100569200 pmid:11673449 fatcat:6ojorjrhpvawjgt4kaxzj3wfoy