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- Unpaywall
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- 0021-9258
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- 10.1074/jbc.273.10.5858
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Abstract
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 10, Issue of March 6, pp. 5858 –5868, 1998 Printed in U.S.A. DNA Double-stranded Breaks Induce Histone H2AX Phosphorylation on Serine 139* (Received for publication, July 25, 1997) Emmy P. Rogakou, Duane R. Pilch, Ann H. Orr, Vessela S. Ivanova, and William M. Bonner‡ From the Laboratory of Molecular Pharmacology, Division of Basic Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892 When mammalian cell cultures or mice are exposed to ilies differ in few if any amino acid residues (5). In contrast, the H2A family includes three subfamilies whose members ionizing radiation in survivable or lethal amounts, novel mass components are found in the histone H2A region of contain characteristic sequence elements that have been con- g, these two-dimensional gels. Collectively referred to as served independently throughout eucaryotic evolution (6, 7). components are formed in vivo by several procedures The three H2A subfamiles are the H2A1-H2A2, the H2AZ, and that introduce double-stranded breaks into DNA. the H2AX; in mammals the H2AZ represents about 10% of the g-Components, which appeared to be the only major H2A complement, the H2AX represents 2–25%, and the H2A1- PO incorpora- novel components detected by mass or H2A2 represents the balance. tion on acetic acid-urea-Triton X-100-acetic acid-urea- In addition, histone species are often modified with phos- cetyltrimethylammonium bromide or SDS-acetic acid- phate and acetate moieties on specific serine and lysine resi- urea-cetyltrimethylammonium bromide gels after dues, respectively, usually near the amino or carboxyl termini. exposure of cells to ionizing radiation, are shown to be A specific role for histone acetylation has been confirmed with histone H2AX species that have been phosphorylated the finding that histone acetylases are transcription factors (8). g-H2AX appears rapidly after specifically at serine 139. Consistent with this is the finding that H4 is acetylated to exposure of cell cultures to ionizing radiation; half-max- higher levels in euchromatin than in heterochromatin (9). Sev- imal amounts are reached by 1 min and maximal eral histone modifications are correlated with chromosome con- amounts by 10 min. At the maximum, approximately 1% densation and mitosis; histone H3 becomes phosphorylated on g-phosphorylated per gray of ion- of the H2AX becomes residue serine 10 (10), and linker histone H1 becomes multiply izing radiation, a finding that indicates that 35 DNA phosphorylated (11). double-stranded breaks, the number introduced by each In this report, we demonstrate that H2AX becomes phospho- base pairs of a mammalian G gray into the 6 3 10 genome, leads to the g-phosphorylation of H2AX distrib- rylated on residue serine 139 in cells when double-stranded breaks are introduced into the DNA by ionizing radiation. One uted over 1% of the chromatin. Thus, about 0.03% of the chromatin appears to be involved per DNA double- of the three H2A subfamilies that has been conserved through- stranded break. This value, which corresponds to about out evolution (12), H2AX comprises 2–10% of the H2A comple- base pairs of DNA per double-stranded break, 2 3 10 ment in mammalian tissues and larger fractions in lower eu- indicates that large amounts of chromatin are involved caryotes where in budding yeast H2AX constitutes virtually all g-H2AX with each DNA double-stranded break. Thus, of the H2A (5). Our finding of a human astrocytoma cell line formation is a rapid and sensitive cellular response to SF268 in which H2AX is 25% of the H2A complement shows the presence of DNA double-stranded breaks, a response that H2AX can be more than 10% of H2A complement in tissue that may provide insight into higher order chromatin culture cells. The sequence that differentiates the H2AX from structures. the other two H2A subfamilies is the C-terminal motif SQ(D/ E)(I/L/Y)-(end). In mammals, the serine in this motif is residue 139, the site of g-phosphorylation. This report is the first dem- In eucaryotes, DNA is packaged into nucleosomes, which are onstration of a unique in vivo function for H2AX, a function in turn arranged in various higher order structures to form that clearly differentiates it from the other H2A species. chromatin (1, 2). The nucleosome, the crystallographic struc- We report that exposure of cell cultures and mice to surviv- ture of which has recently been elucidated (3), is composed of able as well as lethal amounts of ionizing radiation leads to the about 145 bp of DNA and eight histone proteins, two from each induction of g-H2AX. Ionizing radiation has been present dur- of four histone protein families, H4, H3, H2B, and H2A. In ing the evolution of living systems; current background levels, mammals, each histone family is encoded by multiple genes, about 0.5 millisieverts/year, induce on the order of 10 DNA which with few exceptions are expressed in concert with repli- double-stranded breaks each second in the cells of a 50-kg cation (4). The various members of the H4, H3, and H2B fam- mammal. In tissue culture, of every 40 DNA double-stranded breaks introduced per cell by ionizing radiation, approximately * The costs of publication of this article were defrayed in part by the one major karyotypic defect is found (13), defects that may payment of page charges. This article must therefore be hereby marked reflect an unbalanced genome and altered cellular metabolism, “advertisement” in accordance with 18 U.S.C. Section 1734 solely to perhaps leading to cell death or neoplastic progression. indicate this fact. We demonstrate that g-H2AX formation is both a rapid and ‡ To whom correspondence and reprint requests should be addressed: NIH-NCI, Bldg. 37, Rm. 5D17, Bethesda, MD 20892. Tel.: 301-496- sensitive response to ionizing radiation. Half-maximal 5942; Fax: 301-402-0752; E-mail: [email protected]. amounts of g-H2AX are reached by 1 min postirradiation, and The abbreviations used are: bp, base pair(s); Gy, gray; CHO, Chi- nese hamster ovary; TEMED, N,N,N9,N9-tetramethylethylenediamine; BrdUrd, bromodeoxyuridine; AUT, acetic acid-urea-Triton X-100; AUC, acetic acid-urea-cetyltrimethylammonium bromide; DNA-PK, DNA- The HHGRI/NCBI Histone Sequence Database is available on the protein kinase. World Wide Web at http://www.ncbi.nlm.nih.gov/Baxevani/HISTONES. 5858 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. DNA Double-stranded Breaks Induce H2AX Phosphorylation 5859 samples were loaded, electrophoresis was performed at 10 watts over- maximal amounts are reached by 10 min. At the maximum, night. Finished gels were stained in a solution containing acetic acid approximately 1% of the H2AX becomes g-phosphorylated per (5%), ethanol (40%), and Coomassie Brilliant Blue R-250 (0.4%) for 30 Gy of ionizing radiation. This value, which corresponds to min and destained for 30 min in a solution containing acetic acid (5%) about 2 3 10 bp of DNA/double-stranded break, indicates that and ethanol (20%). substantial amounts of chromatin may be involved with each AUC gels were prepared in shells 36 cm wide, 25 cm high, and 1 mm thick. The resolving gel solution contained urea (5 M), acrylamide DNA double-stranded break. Thus, g-H2AX formation is a (18.5%), bisacrylamide (0.11%), acetic acid (1 M), ammonia (0.03 M), rapid and sensitive cellular response to the presence of DNA TEMED (0.5%), and riboflavin (0.0004%). The solution was degassed, double-stranded breaks, a response that may provide insight poured into the shells (leaving 4 cm at the top), overlayered with into higher order chromatin structures. water-saturated butanol, and polymerized between two fluorescent light boxes for 30 min. The stacking gel solution contained urea (5 M), EXPERIMENTAL PROCEDURES acrylamide (5%), bisacrylamide (0.16%), acetic acid (1 M), ammonia Isolation and Labeling of Nuclei—The cell cultures used in this study (0.03 M), TEMED (0.5%), and riboflavin (0.0004%). When the resolving were grown in 10-cm dishes with RPMI 1640 medium containing 10% gels had polymerized, the butanol was removed. The stacking gel solu- fetal calf serum. Nuclei from approximately 10 cells were isolated tion was degassed, poured into the shells (leaving 2 cm at the top), and essentially as described by Whitlock et al. (14). Cell monolayers were polymerized between two fluorescent light boxes for 30 min. washed with cold phosphate-buffered saline. One ml of lysis buffer (10 Regions of interest were excised from the stained first dimension gels mM Tris-HCl, pH 8, 5 mM MgCl , 0.5% Nonidet P-40) was added to each and incubated in a solution containing acetic acid (1 M), ammonia (0.03 of the cell layers, which were scraped into microcentrifuge tubes, and M), and mercaptoethylamine (1%) for 30 min. The pieces were slid into the nuclei were pelleted for2sina microcentrifuge. The histones were the top of a second dimension gel until they rested on the stacking gel. extracted from the pellets with 3 volumes of 0.5 M HCl for 30 min on ice A solution containing 1% melted agarose, acetic acid (1 M), and ammo- and prepared for two-dimensional gel analysis. nia (0.03 M) was poured around and to the top of the inserted sample gel; For labeling studies, nuclear pellets were resuspended in 1 volume of the agarose was allowed to solidify. The reservoir buffer contained TMCD assay buffer (10 mM Tris-HCl, pH 8, 5 mM MgCl ,5mM CaCl , acetic acid (1 M), glycine (0.1 M), and CTAB (0.15%; Sigma H-9151; 2 2 5mM dithiothreitol). One mlof[g- PO ]ATP was added to 9 ml of each hexadecyltrimethylammonium bromide). Electrophoresis was started of the nuclear suspensions, and the mixtures were incubated at 20 °C at 67 milliamps/gel. This was about 12 watts/gel; when the wattage for 20 min. Then 100 ml of ice-cold assay buffer was added to each of the reached 26 watts/gel, the setting was switched to constant wattage at reaction mixtures, which were spun at 1000 rpm in a microcentrifuge 26 watts until the Coomassie Blue migrated to the bottom of the gel. for 5 min; the histones were extracted from the pellets with 3 volumes The total time of electrophoresis was about 7 h. Finished gels were of 0.5 M HCl for 30 min on ice and prepared for two-dimensional gel stained in a solution containing acetic acid (5%), ethanol (40%), and analysis. Coomassie Brilliant Blue R-250 (0.4%) for 2 h and destained in a Exposure of Cell Cultures to Ionizing Radiation—The medium of solution containing acetic acid (5%) and ethanol (20%). These gel reci- CHO, SF268 or other cell cultures was replaced with 10 ml of ice-cold pes were used for all of the AUT-AUC gels presented in this paper medium, and the cultures were exposed to a except those shown in Fig. 1, which contained 18.5% acrylamide in the Cs source at a rate of first AUT dimension; this higher concentration permitted the separa- either 5 or 17 Gy/min in a Shepherd Mark I irradiator. The temperature tion of all of the histone species but with some loss of resolution in the of the medium remained below 8 °C during the irradiation. After irra- H2A region (Fig. 1A versus Fig. 4A). The Coomassie Blue-stained gels diation, the cold medium was replaced with medium at 37 °C, and the were recorded as TIFF images with the Eagleeye II (Stratagene Cloning cultures were returned to the incubator for the times indicated. The Systems), the relevant images were assembled with Paint Shop Pro nuclei and histones were then prepared for analysis. (Jasc, Inc) and Powerpoint (Microsoft), and the figures were printed Exposure of Mice to Ionizing Radiation—DBA/2 mice, 35 days old, with an HP OfficeJet Pro 1150C printer (Hewlett Packard). were irradiated with 200 Gy for 12.5 min at 17 Gy/min or with 3.6 Gy Preparation of Recombinant H2AX—PCR was performed on plas- for 1.5 min at 2.4 Gy/min. The mice were euthanized in a CO chamber mids containing the coding sequences for human H2A1, H2AZ, and at the appropriate times. The livers, about 0.75 g, wet weight, were H2AX (12), maintaining the ATG codon at the 59-end of the coding removed, diced, and homogenized (Polytron, Brinkmann Instruments) in sequence, adding a HindIII site just upstream of the ATG codon and a 5 ml of an ice-cold buffer (10 mM Tris-HCl, pH 7.5, 1 mM MgCl ) for 10 s. convenient restriction site at the 39-end so that the PCR fragments Nonidet P-40 was added to a final concentration of 0.5%, and the suspen- could be cloned in phase into the HindIII site of the pET17xb vector sion was homogenized another 10 s at a slow speed to minimize foaming. (Novagen, Inc.). This procedure permitted the histone species to be The nuclei were pelleted from a 2-ml aliquot of each homogenate by a 2-s expressed as part of fusion proteins. After constructs were checked by spin in a microcentrifuge. Concentrated HCl was added to the pellets to a sequencing, duplex oligonucleotides coding for the formic acid-sensitive final concentration of 0.5 M HCl; histones were extracted for analysis. sequence (Asp-Pro) followed by the nickel-binding sequence His were Two-dimensional Gel Analysis of Histones—Nuclear suspensions 6 6 inserted in phase at the HindIII site. The constructs were expressed in were pelleted for2sina microcentrifuge; the pellets were resuspended bacterial strain BL21(DE3)pLysS (Novagen, Inc.). When expression in 0.5 M HCl and extracted for 30 min on ice. Reaction mixtures con- was maximal, the bacteria were harvested; the pellets were dissolved in taining unbound histone were made 0.5 M in HCl and extracted as 3 volumes of 98% formic acid and incubated at 37 °C overnight, leading above. Acid-insoluble material was pelleted for 5 min in the microcen- to cleavage of the fusion protein species in the (Asp-Pro) region. The trifuge, and the supernatants were removed to other tubes. Powdered formic acid was neutralized with ammonia; the solutions were dialyzed urea was added to each of the supernatants to 8 M, phenolphthalein was versus 10 mM Tris-HCl, pH 7.6, overnight and passed over a nickel added to 0.002%, and concentrated ammonia was added until the solu- column in the appropriate buffer (Novagen, Inc.). The histone species tions became pink. Acetic acid was then added to 1 M, and the samples with their His tags were eluted with an imidazole gradient. The eluted were loaded onto polyacrylamide gels. material was treated with CNOBr to cleave the tagged histone species Histone gels comprise a first acetic acid-urea-Triton X-100 (AUT) at the methionine residue of the initiation codon, lyophilized, dissolved dimension followed by a second acetic acid-urea-cetyltrimethylammo- in the appropriate buffer, and passed through a nickel column to re- nium bromide (AUC) dimension (15). AUT gels were prepared in shells move the His -containing oligopeptides. The histone species were col- 36 cm wide, 45 cm high, and 0.4 mm thick. The resolving gel solution lected in the flow-through and stored at 220 °C. The recombinant contained urea (8 M), acrylamide (12%), bisacrylamide (0.11%), acetic histone species can be reconstituted in vitro into nucleosomes. Histone acid (1 M), ammonia (0.03 M), Triton X-100 (0.5%), TEMED (0.5%), and H2AX mutant constructs were prepared by inserting appropriate du- riboflavin (0.0004%). The solution was degassed, poured into the shells plex oligonucleotides at a SfiI site, unique in the H2AX-pET17xb ex- (leaving 4 cm at the top), overlayered with water-saturated butanol, pression vector and situated in the codon for residue threonine 136. and polymerized between two fluorescent light boxes for 30 min. The Nuclear Extract Labeling of Recombinant H2AX—HeLa nuclear ex- stacking gel solution contained urea (8 M), acrylamide (5%), bisacryl- tracts were prepared from resuspended nuclear pellets by adding 0.1 amide (0.16%), acetic acid (1 M), ammonia (0.03 M), TEMED (0.5%), and volume of 5 M NaCl to the latter. The residual nuclei were pelleted, and riboflavin (0.0004%). When the resolving gels had polymerized, the the kinase extracts were used immediately. Reaction mixes (10 ml) butanol was removed. The stacking gel solution was degassed and contained 1 mlof10 3 TMCD assay buffer; 1 mg of recombinant H2A1, poured into the shells to the top. Sample combs with wells 9 mm wide and 20 mm deep were inserted into the shells, and the gels were polymerized between two fluorescent light boxes for 30 min. The reser- voir buffer contained acetic acid (1 M), and glycine (0.1 M). After the V. S. Ivanova and W. M. Bonner, unpublished observations. 5860 DNA Double-stranded Breaks Induce H2AX Phosphorylation FIG.2. Induction of g-components in cells and mice by nonle- thal amounts of ionizing radiation. A–D, SF268 cell cultures were irradiated with 0 (A), 1.2 (B), 3.6 (C), or 10.8 (D) Gy of ionizing radiation and permitted to recover for 30 min. Histones were extracted and FIG.1. Formation of novel components in cells after ionizing analyzed as described under “Experimental Results.” E–H, mice were radiation. SF268 cell cultures were exposed to 50 Gy from a Cs irradiated, and their liver histones were prepared and analyzed as source at the rate of 17 Gy/min and returned to the 37 °C incubator for described under “Experimental Procedures.” E, unirradiated mouse. F, 30 min. Histones were extracted and analyzed as described under mouse irradiated with 3.6 Gy over 1.5 min and sacrificed 15 min “Experimental Procedures.” The panels present gels containing 18.5% afterward. G, mouse irradiated with 3.6 Gy over 1.5 min and sacrificed acrylamide in the AUT first dimension, while the insets present the 40 min afterward. H, mouse irradiated with 200 Gy over 12 min and H2A portion of gels containing 12% acrylamide in the AUT first dimen- sacrificed 18 min later. The position of the main novel component is sion. A, control. B, gel exposed to 50 Gy. Histone species are noted; noted as g with an arrow when it is present and with a dotted line when uH2A refers to ubiquitinated H2A species. The main novel component it is absent or present in very low amount. In the case of mouse liver, a is noted as g. second arrow denotes a another g-component that migrates faster in the second dimension. H2AX, or mutant H2AX construct; 1 mlof[g- PO ]ATP; and 1 mlof nuclear kinase extract in the appropriate assay buffer. After incubation histone gene products, these gels also permit the separation of for 20 min at 20 °C, the reactions were terminated, and the histone post-translationally modified forms of the histone species (15). proteins were analyzed either by two-dimensional AUT-AUC or by These species, which differ by single charges from each other, one-dimensional SDS gel electrophoresis. generally migrate just behind the parent species in both dimen- RESULTS sions, thus forming a diagonal line (most apparent for H4 in Ionizing Radiation Induces Novel Protein Components Re- Fig. 1, A and B). The charge differences arise most often from solvable on Histone Gels—When mammalian cell cultures are the phosphorylation of serine residues, which adds one nega- exposed to ionizing radiation and the acid-soluble nuclear pro- tive charge to the protein and from the acetylation of lysine teins are analyzed on two-dimensional AUT-AUC gels, novel residues, which removes one positive charge from the protein components that will be referred to as g (Fig. 1, A and B) are (17). H2A (18) and to a lesser extent H2B (19) also have ubiq- found in the H2A region of these gels. In the first AUT dimen- uitin adducts; because of the size of ubiquitin, these adducts sion, histones separate according to peptide length, charge, and migrate in a separate region of the gel (Fig. 1, A and B). the ability to partition onto Triton X-100 micelles. The ability g-Components Are Formed in Cell Cultures and Mice under to bind Triton X-100 micelles is a property of all the known core Nonlethal Conditions—AUT-AUC gel analysis has been per- histone species. Sensitive to single amino acid differences (16), formed on other mammalian cell lines after Cs irradiation, this property enables closely related histone species to be re- including normal human fibroblast IMR90, transformed hu- solved. Since the micelles are uncharged, protein molecules man fibroblast VA13, hamster CHO, human HeLa, and human partitioning onto Triton X-100 micelles are retarded; this par- HL60; all yielded similar results. Thus, g-components are in- titioning and hence the retardation can be modulated by the duced by ionizing radiation in a wide variety of mammalian cells. concentration of urea in the gel. In the second AUC dimension, To help elucidate the physiological relevance of g-compo- the histones separate according to peptide length, charge, and nents, we examined whether or not they are inducible under shape. This combination of separation parameters resolves the survivable conditions. SF268 cultures were exposed to 1.2, 3.6, histones from all other proteins on these two-dimensional gels. or 10.8 Gy of ionizing radiation and permitted to recover for 30 In addition to permitting the resolution of closely related min. g-Components were apparent in all three cases (Fig. 2, DNA Double-stranded Breaks Induce H2AX Phosphorylation 5861 FIG.3. Formation of g-components by other procedures that cause DNA double-stranded breaks. A–D, SF268 cells subjected to the BrdUrd-dye-ultraviolet A light procedure. SF268 cells were grown in the presence of 0.3 mM BrdUrd and 2.5 mM thymidine for 44 h. The medium was removed, and the cell layers were covered with 3 ml of Hoechst dye 33258 dissolved in phosphate-buffered saline (10 mg/ml) at 37 °C for 10 min. The dye was removed, and the cell layers were placed on ice and covered with 1 ml of ice-cold phosphate-buffered saline. The cells were exposed on ice to 10 kJ/m ultraviolet A light from F40T12/BLB bulbs (Sylvania, 365 nm maximum), which required 16.7 min. The cold phosphate-buffered saline was removed, and warm medium was placed on the cell layers, which were returned to the 37 °C incubator for 20 min. Control cultures lacked BrdUrd, dye, or exposure to light. Histones were extracted and analyzed as described under “Experimental Procedures.” E, SF268 cells were incubated with 3 units/ml of bleomycin for 2 h. Histones were extracted and analyzed as described under “Experimental Procedures.” F–H, SF268 cultures were incubated with 10 mM (F)or50 mM (G and H)H O for 30 min at 37 °C. The H O -containing medium was 2 2 2 2 replaced with fresh medium, and the cells were permitted to recover at 37 °C for 30 min. Histones were extracted and analyzed as described under “Experimental Procedures,” except for the positive control (H), which was then exposed to 50 Gy and allowed to recover for 20 min at 37 °C before histone extraction. The position of the main novel component is noted as g with an arrow when it is present and with a dotted line when it is absent 1 5 or present in a very low amount. H2AX S /K refers to H2AX species containing either a phosphate on serine 1 or an acetate on lysine 5. H2AX NO refers to H2AX species with no modification. H2A1s refers to the region where H2A1 isoprotein as well as post-translationally modified species migrate. In panels A and E,a second arrow denotes a another g-component that migrates faster in the second dimension. B–D). While irradiated cells are metabolically active for several BrdUrd and incubated with Hoechst dye 33258 just before days, they may not be able to reproduce. However, cloning irradiation (24). Like ionizing radiation, this method intro- analysis of duplicate SF268 cell cultures showed that there was duces double-stranded breaks as well as single-stranded at least 40% clonal survival at 1.2 Gy and 10% clonal survival breaks into DNA, but unlike the former, the mechanism is at 3.6 Gy (data not shown). Thus, g-components form in cell nonradiolytic. The procedure was found to result in the forma- cultures under survivable conditions. tion of g-components in SF268 cells (Fig. 3, A–D) but only if To determine whether or not the induction of g-components BrdUrd, dye, and light were all present. Since this procedure is a response seen in whole organisms, we exposed mice to leads to the formation of DNA breaks by a nonradiolytic mech- ionizing radiation and extracted the histones from their livers. anism and without hydroxyl radical formation in cells, g-com- Mice were exposed to 3.6 Gy, which is 60% of the 6-Gy LD ponents are not a cellular response directly to ionizing radia- (50% mortality 30 days after exposure) (20 –22); these mice tion or to the presence of hydroxyl radicals, but to the presence would be expected on average to have a life span shortened by of DNA breaks. This result was substantiated by the presence only 10 –15% (23). g-Components were apparent 15 (Fig. 2F) of g-components when SF268 cell cultures were incubated and 40 min (Fig. 2G) after exposure to 3.6 Gy. g-Components with bleomycin (25), a compound that also introduces double- were more abundant when mice were exposed to 200 Gy (Fig. and single-stranded breaks into cellular DNA by a nonradio- 2H), which kills mice within several hours. Thus, g-components lytic mechanism (Fig. 3E). form in living organisms at both nonlethal and lethal amounts While the above described procedures introduce DNA dou- of ionizing radiation. ble- and single-stranded breaks without hydroxyl radical for- g-Components Are Induced by DNA Double-stranded mation, H O produces hydroxyl radicals and DNA single- 2 2 Breaks—Since the formation of g-components appeared to be a stranded breaks, as does ionizing radiation, but does not widespread cellular reaction to ionizing radiation among mam- produce significant amounts of DNA double-stranded breaks mals, it is relevant to determine whether the cell cultures are because the cellular distribution of the hydroxyl radicals differs responding directly to the ionizing radiation or to a particular between the two agents (26). With H O , radicals are generated 2 2 type of cellular damage induced by the ionizing radiation. Ion- homogeneously throughout the cell as contrasted to the heter- izing radiation introduces many different kinds of damage into ogeneous distribution found with ionizing radiation. Incuba- cells, directly by collision with atoms of biological molecules tion of SF268 cell cultures with 10 mM (Fig. 3F)or50 mM (Fig. and indirectly by collision with water molecules. The latter 3G)H O for 30 min did not lead to detectable formation of 2 2 generates free radicals, of which the most abundant is the g-components, although these cultures were still able to form hydroxyl radical. Ionizing radiation produces high local concen- g-components after exposure to ionizing radiation (Fig. 3H). trations of hydroxyl radicals that, if located next to a DNA These concentrations are damaging to cells; incubation of CHO molecule, may produce locally multiply damaged sites (13) cultures with 50 mM H O for 30 min at 37 °C was found to 2 2 containing alterations of the base and sugar residues and result in approximately 95% clonal lethality (27). Another breaks of one or both strands of the DNA double helix. agent that damages cellular DNA primarily by introducing Several agents and procedures that do or do not introduce single-strand lesions (26) is ultraviolet C light. When SF268 double-stranded breaks into the DNA in cells were examined cell cultures were irradiated with 1, 3, 10, 30, or 100 J/m of (Fig. 3). Cellular DNA can be sensitized to form DNA double- ultraviolet C radiation, amounts of radiation that cover the and single-stranded breaks upon irradiation with ultraviolet A range from little if any cellular effect to complete lethality, no light (350 nm) when cell cultures are grown in the presence of g-components were detected after a 30-min recovery (data not 5862 DNA Double-stranded Breaks Induce H2AX Phosphorylation FIG.5. Phosphorylation of g-components: SDS-AUC gels. SF268 FIG.4. Phosphorylation of g-components: AUT-AUC gels. cells were grown almost to confluence on 10-cm dishes. A, first dimen- SF268 cells were grown almost to confluence on 10-cm dishes. A and C, sion SDS gel used for the two-dimensional gel presented in panel B. B one dish was incubated for 30 min at 37 °C with 5 ml of PO -free RPMI and D, one culture was incubated for 30 min at 37 °C in 5 ml of PO -free 1640 medium with 10% fetal calf serum and containing 1 mCi of PO RPMI 1640 medium with 10% fetal calf serum and containing 1 mCi of (1000 mCi/mmol; NEN Life Science Products). B and D, a duplicate dish PO (1000 mCi/mmol; NEN Life Science Products). C and E, a dupli- received 50 Gy on ice and then was incubated as above. Histones were cate culture received 50 Gy on ice and then was incubated as above. extracted and analyzed as described under “Experimental Procedures.” Histones were extracted and analyzed as described under “Experimen- A and B, Coomassie Blue stain. C and D, autoradiograph. The position tal Procedures.” A and B, Coomassie Blue stain. C and D, autoradio- of the main novel component is noted as g with an arrow when it is graph. The position of the main novel component is noted as g with an present and with a dotted line when it is absent or present in a very low arrow when it is present and with a dotted line when it is absent or amount. The dotted boxes outline the ubiquitinated H2A region in present in a very low amount. The ubiquitinated H2A region shown in panels A–D, and in panels C and D, a longer exposure of the boxed area panels D and E is from a longer exposure. The other nomenclature is is reproduced in the upper right corner. The other nomenclature is explained in the legends to Figs. 1 and 3. explained in the legends to Figs. 1 and 3. shown). Thus, it is the DNA double-stranded break from ion- nated to the same extent as the H2A species when detected by izing radiation that is responsible for g-component formation. mass (inside the dotted rectangles, Fig. 4, A and B) and by label g-Components Are Phosphorylated H2A Derivatives—The (inside the dotted rectangles, Fig. 4, C and D). These results AUT-AUC gels shown in Fig. 1 were prepared with a 18% first from AUT-AUC gels indicate that the g-components are phos- dimension AUT gel to resolve all histone species. The AUT- phorylated H2A derivatives. AUC gels shown in the other figures were prepared with a 12% To obtain more information about the identity of g-compo- first dimension AUT gel to optimize the separation of g-com- nents, similarly prepared samples were subjected to SDS-AUC ponents; however, H4, H2B, and several of the H3 isoforms gel analysis. In SDS gels (Fig. 5A), proteins separate primarily migrate at the buffer front in this dimension and thus are according to size. H2AX migrates almost coincidentally with separated only in the second. g-Components were obtained H3 in SDS gels, but the two are resolved on AUC gels (Fig. 5, when SF268 cultures were exposed to 50 Gy of ionizing radia- B and C). g-Components were not apparent on SDS-AUC gels tion and returned to a 37 °C incubator for a 30-min recovery by Coomassie Blue stain (Fig. 5, B and C) but were apparent 32 32 period (Figs. 1B and 4B). When PO was included in the when the PO label was detected (Fig. 5, D and E), because 4 4 medium during the recovery period, g-components became ra- g-components migrate coincidentally with H3 in both SDS and dioactively labeled (Fig. 4D). The pattern of the g-components AUC gels, but not in AUT gels. Thus, to separate g-components appeared to mimic the pattern of the H2AX species with its from other proteins it is necessary to utilize AUT gels to resolve previously characterized modified forms (17). H2AX, as well as the histone species followed by AUC gels to resolve the g-com- H2A1 and H2A2, can be phosphorylated on residue serine 1 ponents from the known modified forms of H2AX. 1 5 and/or acetylated on residue lysine 5 (noted as H2A S /K on AUT-AUC and SDS-AUC gels contain many other proteins, Fig. 4A) and/or ubiquitinated on residue lysine 119 (uH2A, only some of which are detectable by Coomassie Blue stain. Of inside the dotted rectangle in Fig. 4A). The phosphorylation of components that were visible in these experiments either by 32 32 serine 1 accounts for the presence of PO label found in the stain or by PO labeling, the g-components were by far the 4 4 1 137 H2A region in the control cultures (noted as H2A S in Fig. 4B). most heavily labeled and the only major ones induced by Cs After Cs irradiation, labeling on H2A serine 1 was de- irradiation (Figs. 4 and 5). It is also apparent that either with creased, while the g-components became heavily labeled (Fig. or without Cs irradiation of these cultures, there was no 4, C and D). Note that the g-components appear to be ubiquiti- significant PO incorporation into H3 (Fig. 4, C and D) or into 4 DNA Double-stranded Breaks Induce H2AX Phosphorylation 5863 the H2B and H4 species (Fig. 5, D and E). H2AX do not resolve from each other in either of these systems. g-Components Can Be Formed in Vitro—We had found that However, with the multiplicity of g-components (Fig. 6B), the PO -labeled g-components can be generated in vitro with relationship of g-components to H2AX appears even more com- isolated nuclei and in nuclear extracts with recombinant pelling. More components are visible for two reasons. The first H2AX. These findings stemmed from reports that a histone, is the higher specific activity attainable with in vitro radioac- originally identified as H3 from SDS gels (14, 28, 29) and later tive labeling. The second is the growth of the cells in 5 mM as H2AX (30), was the only protein labeled to a significant sodium butyrate for several hours before harvest, a condition extent when isolated nuclei were incubated with [g- PO ]ATP. 4 that leads to increased acetylation of histone (14). Under these The report of H2AX as the labeled histone species (30) utilized in vitro labeling conditions, mass amounts of g-components are an AUT-SDS gel system and thus did not resolve g-components not expected, since there is no source of bulk phosphate and from H2AX, since, as shown in Figs. 4 and 5, g-components and ATP (Fig. 6, A and B). The in vitro labeling of nuclei is also neither dependent on nor increased by exposure of the nuclei to ionizing radiation; this finding is possibly due to the introduc- tion of DNA double-stranded breaks during the centrifugation steps used to isolate nuclei. The kinase activity can be extracted with 0.35 M NaCl from nuclei and is capable of phosphorylating histones in solution (14). We prepared recombinant H2A1, H2AZ, and H2AX spe- cies and examined their ability to be phosphorylated with 0.35 M NaCl extracts. Only H2AX could be labeled with [g- PO ] ATP to a significant extent with these extracts; in addition, the labeled material migrated primarily as g-components (Fig. 6, C and D). g-Components did not result when recombinant H2AX was labeled with protein kinase C; the radioactive material migrated on AUT-AUC gels at the position of H2AX phospho- rylated on residue serine 1 (data not shown). Thus, these in vitro experiments confirm that g-components are H2AX derivatives. g-Component Is H2AX-phosphorylated on Serine Residue 139 —The finding that recombinant H2AX forms PO -labeled g-components enabled us to identify the site of modification(s) as well as the recognition parameters of the relevant kinase. The strategy to localize the site of g-phosphorylation utilizing recombinant H2AX is presented in Fig. 7, which displays the sequences of H2A1 and H2AX. The two sequences are almost identical up to residue lysine 119, but differ both in sequence and length in the C-terminal region. As mentioned previously, the serine at position 139 is the prime candidate for the site of g-phosphorylation, since it has been conserved throughout evo- lution in at least one H2A species in each animal species FIG.6. g-component formation in vitro. A and B, nuclei prepared examined. from SF268 cells were incubated with [g- PO ]ATP as described under “Experimental Procedures.” The cell cultures were incubated for 3 h H2AX protein has glutamic acid residues at positions 92 and before nuclear isolation with 5 mM sodium butyrate to increase in vitro 141 with no intervening aspartic acid and arginine residues. labeling (14). C and D, phosphorylation of recombinant H2AX by nu- Digestion with the protease StaphV8, which cleaves at aspartic clear extract as described under “Experimental Procedures.” Carrier and glutamic acid residues, would be expected to yield a histone from SF268 cells irradiated with 50 Gy was added before loading the gel. A and C, Coomassie Blue stain. B and D, autoradio- polypeptide 49 residues long, while clostripain digestion would graphs. The position of the main novel component is noted as g with an be expected to generate a 57-residue C-terminal fragment that arrow when it is present and with a dotted line when it is absent or subsumes the 49-residue StaphV8 polypeptide (Fig. 7). In con- 1 5 present in a very low amount. H2AX S &K refers to H2AX species trast, trypsin digestion would be expected to generate small containing a phosphate on serine 1 and an acetate on lysine 5. The other nomenclature is explained in the legends to Figs. 1 and 3. C-terminal fragments. This C-terminal region of the H2AX FIG.7. Sequences of H2A1, H2AX, and recombinant H2AX constructs. Light and medium lines indicate the lengths of peptides formed, respectively, by clostripain and StaphV8 cleavage of H2AX; the short heavy line indicates the H2AX C-terminal peptide derived from trypsin digestion. The identities of the five proven and putative H2AX modifica- tion sites discussed in this work are shown with the affected amino acid resi- due. The bacterial expression plasmid en- coding human H2AX was altered by the insertion of oligonucleotides coding for ATPWER (H2AX-c22) or AAPWER (H2AX-c23) at a SfiI site of the human H2AX sequence by standard cloning tech- niques, and recombinant proteins were produced as described under “Experimen- tal Procedures.” 5864 DNA Double-stranded Breaks Induce H2AX Phosphorylation sequence is the only region in any core histone that yields size of 57 residues (Fig. 7), while in construct H2AX-c22 (Fig. 7) similarly sized large peptides with clostripain and StaphV8. almost all of the label was found in a small peptide consistent To confirm that g-components contain phosphate in the pre- with the predicted size of 8 residues (Fig. 8B). Digestion with dicted region, natural PO -labeled g-component was digested StaphV8 also yielded new small peptides with construct H2AX- with clostripain, StaphV8, and trypsin. Digestion with the first c22 as predicted, substantiating the result with clostripain. two resulted in large similarly sized radioactive fragments, StaphV8 digestion would also be expected to remove the ter- while trypsin digestion resulted in a small one (Fig. 8A). Next, minal tyrosine, indicating that the phosphorylation takes place recombinant H2AX labeled with [g- PO ]ATP and nuclear on the peptide ATQASQE (Fig. 8B). In construct H2AX-c23, an kinase was digested with the same enzymes and was found to alanine residue replaces the threonine residue to yield a tryptic yield the same pattern of fragments (Fig. 8B, the lanes noted as peptide AAQASQEY; that this construct is phosphorylated to a 0 under none (N), trypsin (T), clostripain (C), and StaphV8 (S)) similar extent as is H2AX-c22 indicates that threonine residue to that obtained with the natural g-component. Thus, g-com- 136 is not a major phosphorylation site but may be a secondary ponent is a form of H2AX phosphorylated in the C-terminal site. Histones do not contain tryptophan, thus N-bromosuccin- region. imide treatment under conditions that cleave proteins only at To determine which residue is involved in the g-phosphoryl- tryptophan residues does not cleave construct H2AX-c0 (Fig. ation of H2AX, we prepared two recombinant H2AX derivatives 8B, N); however, construct H2AX-c22 is cleaved under these with altered amino acid sequences (Fig. 7) that would provide conditions to yield a small labeled peptide consistent with the new cleavage sites at predicted positions for the enzymes men- expected size (Fig. 8B, N). These results demonstrate that tioned above. Clostripain digestion of the parent H2AX-c0 con- g-components are H2AX species phosphorylated on serine 139. struct labeled with nuclear extract and [g- PO ]ATP yielded a Recognition Site for g-Phosphorylation—A major advantage large but less than full-length labeled peptide consistent with a of using recombinant H2AX constructs to determine the site of phosphorylation is that they also allow us to determine some of the recognition parameters of the kinase for g-phosphorylation. To do so, a second set of recombinant H2AX derivatives were prepared with various alterations in the C-terminal sequence (Table I). Substituting serine 139 with leucine decreased activ- ity of the construct to 9.6% of the H2AX control, an expected finding, since this is the site of phosphorylation; however, glu- tamine 140 appeared to be just as essential (H2AX-c9). Neither of these constructs gave activities as low as that of H2A1, however (compare H2AX-c1 and H2AX-c9 with H2A1-wt), pos- sibly due to a small amount of phosphorylation at threonine 136 in the H2AX constructs. Supporting this possibility is the finding that threonine did appear to substitute well for serine 139 (H2AX-c8). Glutamate 141 appeared to be relatively unim- portant to the specificity (H2AX-c7). Changes in length that placed serine 139 either closer to or farther from the C termi- nus appeared to be of lesser importance (H2AX-c2, -c3, and -c6). On the other hand, that there may be other determinants of FIG.8. Peptides of g-H2AX. A, natural g-component labeled with specificity was indicated by the low activity of constructs [g- PO ]ATP in whole nuclei was isolated from the gel, electroeluted, H2AX-c4 and c5, in which the SQ motif was present but in dialyzed against water, and freeze-dried. The material was dissolved in different backgrounds. These results show that the SQ se- the appropriate buffers and incubated with water (U), trypsin (T), quence is an important determinant for g-phosphorylation of clostripain (C), or staphylococcus V8 (S) (all from Promega). B, recom- binant protein constructs H2AX-wt, -c22, and -c23 were labeled as H2AX. described under “Experimental Procedures” and incubated as above These findings allow us to assign structures to each of the and with N-bromosuccinimide (Sigma). All digests were mixed with one modified H2AX species. Fig. 9A presents an enlargement of the volume of 2 3 SDS sample buffer, boiled for 5 min, and analyzed by H2AX and g-component region from the gel presented in Fig. electrophoresis on 30% acrylamide gels containing SDS. The wet gels were immediately exposed to film at room temperature. 6B. A grid is superimposed over the pattern of H2AX and TABLE I Activity of altered H2AX analogues as nuclear kinase substrates Constructs were prepared and assayed as described under “Experimental Procedures.” SDS gels were stained with Coomassie Blue, and the radioactivity was assayed with the Betagen. Construct Sequence Alteration Percentage of wild type Controls H2AX-wt (133 residues)-katqasqey-COOH H2AX control 100 H2A1-wt (132 residues) H2A1 control 2.2 Single residue substitutions H2AX-c1 (133 residues)-katqaLqey-COOH Ser-139 to Leu 9.6 H2AX-c8 (133 residues)-katqaTqey-COOH Ser-139 to Thr 52.0 H2AX-c9 (133 residues)-katqasNey-COOH Gln-140 to Asn 7.2 H2AX-c7 (133 residues)-katqasqNy-COOH Glu-141 to Asn 49.0 Changes in length H2AX-c3 (133 residues)-katqasqeyGK-COOH Add GK to C terminus 76.0 H2AX-c6 (133 residues)-katqasqe-COOH Delete C-terminal Tyr 42.0 H2AX-c2 (133 residues)-katqasq-COOH Delete C-terminal EY 22.0 Multiresidue changes H2AX-c5 (21 residues)-katqasqey-COOH Delete N-terminal 112 residues 2.9 H2AX-c4 (133 residues)-katqalhhksqtk-COOH Rat testes C-terminal hybrid 4.6 DNA Double-stranded Breaks Induce H2AX Phosphorylation 5865 FIG.9. Assignment of g-H2AX deriv- atives to particular structures. A,an enlargement of the in vitro labeled mate- rial presented in Fig. 6B with a grid su- perimposed. B, the same grid with the modified forms present in each square. 1/5 refers to H2AX species modified in the 1- or the 5-position, as presented in Fig. 7. 1&5 refers to H2AX species modi- fied in both positions. Modifications at po- sitions 136 and 139 are likewise noted. FIG. 11. Relative amount of g-H2AX at various times after ex- posure to ionizing radiation: quantitation. The H2AX components on the TIFF images presented in the upper panel were quantitated with ImageQuant software version 3.3 (Molecular Dynamics) without any contrast or brightness enhancement. The open circle indicated by the arrow (lower left) shows the percentage of g-H2AX measured in unirra- FIG. 10. Relative amount of g-H2AX at various times after ex- diated cells. The open and filled symbols denote separate experiments. posure to ionizing radiation: gels. Cultures of hamster CHO cells were exposed to 200 Gy (12 min at 18 Gy/min) at 4 – 8 °C. The cold medium was replaced by medium at 39 °C (except sample A, which was diation, hamster CHO cell cultures were irradiated on ice with harvested immediately), and the cultures were allowed to recover for 200 Gy from a Cs source, rapidly returned to 37 °C, and 20s(B), 1 min (C), 3 min (D), 9 min (E), 15 min (F), 30 min (G), 60 min (H)or90min(I). Histones were extracted and analyzed as described allowed to recover for various times. g-H2AX did not form in under “Experimental Procedures.” TIFF images of the Coomassie Blue- cell cultures on ice but was visible 20 s after returning them to stained gels of histones H2A1 and H2AX were recorded with the Eagle- 37 °C (Fig. 10B). g-H2AX increased in amount within 10 min to eye II (Stratagene Cloning Systems). The position of the main novel more than 50% of the total H2AX (Fig. 10, C–F), remained at component is noted as g with an arrow when it is present and with a that level until about 30 min postirradiation (Fig. 10G), and dotted line when it is absent or present in a very low amount. then decreased over a period of hours (Fig. 10, H–I). Densito- g-components; the same grid is reproduced in Fig. 9B with metric analysis of the amounts of H2AX and g-H2AX from the modification sites, as noted in the H2AX sequence (Fig. 7), gels presented in Fig. 10 is shown in Fig. 11. The data form a assigned to each of the nine forms. The pattern of ubiquitinated smooth curve with a rapid rise and a slower decrease. While the forms is identical to that of the nonubiquitinated forms (Fig. maximum is reached at 9 –30 min, half the maximum is 6B, inset), except that each H2AX molecule also contains a reached in 1 min. Although these experiments were performed ubiquitin modification on residue lysine 119. These assign- with large amounts of ionizing radiation to obtain a significant ments also provide an explanation for the different relative signal at short times, similar time courses are observed at amounts of a second faster g-component seen in mouse and lower amounts (data not shown). However, with mice at non- human cells (second arrow in Fig. 2, F–H, and Fig. 3, A and E). lethal amounts of ionizing radiation, quantitation of the images Mouse H2AX contains a serine residue at position 136 as well presented in Fig. 2, E–G, yielded values of approximately 2% as at position 139 (31), while the human form has a threonine g-H2AX at 15 min (Fig. 2F), 5% at 40 min (Fig. 2G), and less residue at position 136. If serine is the preferred substrate for than 2% at 70 min (data not shown), a time course consistent the kinase, then mouse g-H2AX might be expected to contain with that shown in Fig. 11. Because of these results, 30 min more of a doubly g-phosphorylated H2AX. was chosen as the optimum recovery time when other param- g-H2AX Forms within Seconds after Exposure of Cells to eters were studied. Incubation of the irradiated cultures with Ionizing Radiation—Ionizing radiation can be delivered in pre- the protein synthesis inhibitor cycloheximide (100 mg/ml) did cisely measured amounts and time periods. In addition, its not prevent g-phosphorylation (data not shown), indicating effects on cells have been intensively investigated qualitatively that any proteins necessary for g-modification are already pres- and quantitatively; ionizing radiation from Cs results in 35 ent. Likewise, inhibiting DNA replication with hydroxyurea DNA double-stranded breaks/G genome in mammalian cells (10 mM) had no effect on g-H2AX formation. (13, 26, 32). These parameters enable us to determine the g-H2AX Formation Is Proportional to the Amount of Radia- kinetics and stoichiometry of g-H2AX formation. To determine tion—When hamster CHO cell cultures were subjected to dif- how quickly g-components form after exposure to ionizing ra- ferent amounts of ionizing radiation, the fraction of g-H2AX 5866 DNA Double-stranded Breaks Induce H2AX Phosphorylation FIG. 12. The relative amount of g-H2AX is proportional to the FIG. 13. The relative amount of g-H2AX is proportional to the amount of radiation: gels. Cultures of hamster CHO cells were amount of radiation: quantitation. Filled circles, the H2AX compo- exposed to 0 (A), 12.5 (B), 25 (C), 50 (D), 100 (E), or 200 (F)Gyof nents on the TIFF images presented in the upper panel along with a ionizing radiation and permitted to recover at 37 °C for 30 min. His- duplicate set of cultures exposed separately were quantitated with tones were extracted from the cultures and analyzed as described. TIFF ImageQuant software version 3.3 (Molecular Dynamics) without any images of the Coomassie Blue-stained gels of histones H2A1 and H2AX contrast or brightness enhancement. Open circles and squares, quanti- were recorded with the Eagleeye II (Stratagene Cloning Systems). The tation of two similar experiments performed with human SF268 cell position of the main novel component is noted as g with an arrow when cultures allowed to recover for 30 min. The arrows denote the data it is present and with a dotted line when it is absent or present in a very points from the 1.2- and 3.6-Gy samples shown in Fig. 2, B and C. low amount. the chromatin but is likely to be randomly distributed among was found to increase with the amount of radiation (Fig. 12, the nucleosomes. Supporting this assumption is the fact that in A–F). Densitometric analysis of the data is presented in Fig. 13. lower eucaryotes most or all of the H2A is H2AX (5). If H2AX is The initial slope of the curve for CHO cells (filled circles) randomly distributed throughout the chromatin, then the frac- indicates that about 1.0% of the H2AX complement became tion of g-H2AX is a measure of the fraction of the chromatin g-modified per Gy of radiation. This value may be underesti- and hence of the DNA that is involved per Gy. Thus, the mated because it assumes that all of the relevant H2AX is in simplest explanation for these findings is that a similar region the g-modified state simultaneously. On the other hand, the of the chromatin is involved per Gy irrespective of the cell line. H2AX in hamster CHO cultures receiving 200 Gy became 67% One Gy of ionizing radiation causes 35 DNA double-stranded g-modified (Fig. 11F) after a 30-min recovery, indicating that breaks/G genome, which is 6 3 10 bp of DNA in mammalian the maximal value of g-H2AX modification is probably no more cells. If 1% of the chromatin is involved per 35 DNA double- than 1.5% per Gy for CHO cells. Results with the normal stranded breaks, then 0.03% is involved in each. 0.03% of 6 3 9 6 human fibroblast line IMR90 were found to be very similar to 10 bp is 1.8 3 10 bp. Thus, one of the intriguing implications those obtained with CHO cells (data not shown). of these findings is that megabase regions of chromatin appear Densitometric results are also shown for SF268 cultures to be involved in each DNA double-stranded break. (Fig. 13, open symbols). g-H2AX formation in this cell line DISCUSSION appears to be more sensitive to ionizing radiation, perhaps related to its high relative content of H2AX. Note that with H2AX becomes phosphorylated on serine 139 rapidly and SF268, the amount of g-H2AX induced per Gy at the survivable extensively after exposure of mammalian cell lines and mice to amounts of radiation, 1.2 and 3.6 Gy (Fig. 2, B and C; Fig. 13, various procedures that lead to the formation of DNA double- arrows) is similar to that induced at the low nonsurvivable stranded breaks. g-H2AX formation begins within seconds af- amounts, indicating that g-H2AX formation seen under lethal ter exposure to ionizing radiation and rapidly passes through a conditions is the same process as that seen under survivable half-maximal value at 1 min to a maximal value at 9 –30 min. conditions. The efficiency of g-H2AX formation is similar in DNA double-stranded breaks are repaired by various mecha- mice. At 40 min postirradiation (Fig. 2G), formation of g-H2AX nisms in mammalian cells. One pathway involves the DNA-PK was approximately 1.4% per Gy, results comparable with those complex that is defective in scid mice (33). Evidence for a found in cell culture (Fig. 13). second DNA double-stranded break repair system has recently g-H2AX Modification and the Relative Abundance of H2AX— been reported (34); this system, which functions during G , Previous data have shown that the fraction of H2AX converted appears to be normal in scid cells. H2AX is a substrate for to g-H2AX forms is similarly dependent on the amount of phosphorylation by DNA-PK in vitro (35), and we found that ionizing radiation in both CHO cells and normal human IMR90 purified DNA-PK g-phosphorylated purified H2AX in vitro fibroblasts. Both these cell lines contain H2AX as 9 –10% of the (data not shown). However, when we examined several of the total H2A complement. However, we have investigated g-H2AX cell lines that are known to be deficient in DNA-PK, either in formation in cell lines with H2AX comprising as little as 2.4% the catalytic subunit or in the Ku subunits (36, 37), neither the and as much as 25% of the total H2A complement (Table II). C.B-17-SCID mouse cell line (33), nor the V3 hamster mutant Since the number of DNA double-stranded breaks introduced line (38), nor the ICR-SCID mouse (39) exhibited a noticeable per Gy per unit of chromatin is the same irrespective of cell deficit in g-H2AX formation after exposure to ionizing radia- lineage (13), this variation enables us to ask whether there is a tion. With the above mentioned mutant cell lines and mice, it is constant number or a constant percentage of g-H2AX molecules also possible that residual DNA-PK activity is present, since formed per DNA double-stranded break. The data in Table II these are not knockouts. A cell line from the Ku80 knockout are consistent with a similar percentage but not a similar mouse (40) was donated by Gloria Li. This line also showed number of H2AX molecules being g-phosphorylated per DNA normal g-H2AX formation. Thus, g-H2AX formation could re- double-stranded break; each Gy of ionizing radiation leads to sult from another DNA double-stranded break repair system the g-phosphorylation of about 1–2% of the H2AX irrespective that does not utilize DNA-PK or from a step upstream of cs of whether the H2AX accounts for 2.5 or 25% of the total H2A DNA-PK action. cs complement. With the variation in its relative abundance, Human cell line M059J, a mutant line lacking DNA-PK H2AX is unlikely to be localized to certain specific regions of protein (41) donated by Joan Turner did show a substantial DNA Double-stranded Breaks Induce H2AX Phosphorylation 5867 TABLE II Constant percentages, not numbers, of H2AX molecules are g-modified per Gy The stained H2A2, H2A1, and H2AX species on two-dimensional gels were recorded as TIFF images and quantitated with ImageQuant software version 3.3. The g-H2AX/H2AX ratio was determined 30 min after exposing the cell cultures to 25 Gy. The following conversion factors and 9 6 assumptions were used. 1) The mammalian G genome contains 6 3 10 bp of DNA, hence about 30 3 10 nucleosomes (200 bp/nucleosome) and 60 3 10 H2A molecules (2 molecules/nucleosome). 2) 25 Gy induces about 875 DNA double-stranded breaks per G genome. 3) H2AX is randomly distributed in the chromatin. Cell type H2AX/total H2A g-H2AX/total H2AX No. of H2AX/cell No. of g-H2AX/cell No. of g-H2AX/dsb g-H2AX/dsb bp of DNA/dsb %% % 6 6 6 VA13 2.6 28 1.6 3 10 0.45 3 10 530 0.033 2.0 3 10 6 6 6 HeLa 2.4 30 1.4 3 10 0.45 3 10 490 0.035 2.1 3 10 6 6 6 IMR90 9.8 30 5.9 3 10 1.7 3 10 2100 0.035 2.1 3 10 6 6 6 CHO 9.4 34 5.6 3 10 1.9 3 10 2240 0.040 2.4 3 10 6 6 6 SF268 25 50 15 3 10 7.5 3 10 8800 0.059 3.5 3 10 deficiency in g-H2AX formation. Fifteen min after 200 Gy, the exterior of the nucleosome would make H2AX residue serine control M059K line converted over 60% of its H2AX to g-forms, 139 easily accessible to kinases. If kinases tracked along the a value similar to that obtained in other mammalian cell lines. DNA about equal distances from a DNA double-stranded break In contrast, the mutant M059J line under the same conditions in the various cell lines before falling off or encountering a contained no more than 25% g-H2AX. Human cells contain barrier, then about equal percentages of g-H2AX would be about 10 times as much DNA-PK activity as do rodent cells, formed irrespective of relative H2AX content. This model pro- indicating that there may be important differences in these vides an explanation why a constant percentage and not a enzyme systems of the two groups. constant number of the H2AX molecules are g-phosphorylated The locus for the ataxia-telangiectasia defect is 11q23, close per Gy in various cells. to that of the H2AX. However, three ataxia-telangiectasia cell It is not necessary to postulate chromatin structures of lines from complementation groups A, C, and D were found to megabase dimensions, but there is evidence supporting chro- contain H2AX and in addition showed no significant deficit in matin structures of this size. Yokota et al. (43), measuring the g-H2AX formation after exposure to ionizing radiation (data physical distance between probes of known separation along not shown). Thus, the ataxia-telangiectasia kinase (42) is not the DNA in interphase nuclei, reported a discontinuity between responsible for g-H2AX formation after irradiation. The defects the physical and genomic distances at about 2 3 10 bp, indi- in other human genetic radiosensitive diseases are located on cating that an underlying chromatin structure of this size may other chromosomes, suggesting that these diseases are not due be present. In addition, Yunis (44) using preparations of mid- to defective H2AX protein (34). H2AX knockout cell lines and prophase human chromosomes stained with Giemsa was able mice will help determine the role of g-H2AX formation in cel- to discern about 2000 bands/haploid complement; those values lular metabolism. We are currently investigating possible re- yield an average size of 1.5 3 10 bp/band. lationships between the kinase responsible for g-H2AX forma- In a second type of hypothesis, the DNA double-stranded tion and other kinases. break would still be the initiating point of the g-H2AX forma- At the time of maximal modification, H2AX on 1–2% of the tion, but the activity would diffuse away from the break in chromatin is g-modified. Since each Gy causes 35 DNA double- three-dimensional space. Thus, H2AX molecules on strands of stranded breaks/6 3 10 bp, this is an amount of chromatin chromatin near the DNA double-stranded break would become equivalent to 1.8 –3.5 3 10 bp of DNA/double-stranded break. g-phosphorylated even if those strands were on different chro- While this is a strikingly large amount of chromatin, the DNA mosomes. In a third type of hypothesis, H2AX molecules at double-stranded break is a serious lesion that often leads to random throughout the nucleus would become phosphorylated chromosomal abnormalities; of the 35 DNA double-stranded in a manner dependent on the amount of radiation. Determin- breaks/genome per Gy, approximately one will result in a vis- ing the spatial relationship between the triggering lesion and ible chromosomal abnormality, and more may be present that the g-H2AX will be useful in elucidating their functional rela- are not detectable by cytological analysis (13). Thus biological tionship. Antibodies specific to g-H2AX will be useful in deter- systems may have evolved very sensitive detection systems for mining the spatial characteristics of the response. DNA double-stranded breaks; g-H2AX formation may function Acknowledgments—We gratefully acknowledge Dr. Kurt Kohn for in such a system. continuing support. 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Journal
Journal of Biological Chemistry – Unpaywall
Published: Mar 1, 1998