DNA Double-stranded Breaks Induce Histone H2AX Phosphorylation on Serine 139

When mammalian cell cultures or mice are exposed to ionizing radiation in survivable or lethal amounts, novel mass components are found in the histone H2A region of two-dimensional gels. Collectively referred to as γ, these components are formed in vivo by several procedures that introduce double-stranded breaks into DNA. γ-Components, which appeared to be the only major novel components detected by mass or 32PO4incorporation on acetic acid-urea-Triton X-100-acetic acid-urea-cetyltrimethylammonium bromide or SDS-acetic acid-urea-cetyltrimethylammonium bromide gels after exposure of cells to ionizing radiation, are shown to be histone H2AX species that have been phosphorylated specifically at serine 139. γ-H2AX appears rapidly after exposure of cell cultures to ionizing radiation; half-maximal amounts are reached by 1 min and maximal amounts by 10 min. At the maximum, approximately 1% of the H2AX becomes γ-phosphorylated per gray of ionizing radiation, a finding that indicates that 35 DNA double-stranded breaks, the number introduced by each gray into the 6 × 109 base pairs of a mammalian G1 genome, leads to the γ-phosphorylation of H2AX distributed over 1% of the chromatin. Thus, about 0.03% of the chromatin appears to be involved per DNA double-stranded break. This value, which corresponds to about 2 × 106 base pairs of DNA per double-stranded break, indicates that large amounts of chromatin are involved with each DNA double-stranded break. Thus, γ-H2AX formation is a rapid and sensitive cellular response to the presence of DNA double-stranded breaks, a response that may provide insight into higher order chromatin structures. When mammalian cell cultures or mice are exposed to ionizing radiation in survivable or lethal amounts, novel mass components are found in the histone H2A region of two-dimensional gels. Collectively referred to as γ, these components are formed in vivo by several procedures that introduce double-stranded breaks into DNA. γ-Components, which appeared to be the only major novel components detected by mass or 32PO4incorporation on acetic acid-urea-Triton X-100-acetic acid-urea-cetyltrimethylammonium bromide or SDS-acetic acid-urea-cetyltrimethylammonium bromide gels after exposure of cells to ionizing radiation, are shown to be histone H2AX species that have been phosphorylated specifically at serine 139. γ-H2AX appears rapidly after exposure of cell cultures to ionizing radiation; half-maximal amounts are reached by 1 min and maximal amounts by 10 min. At the maximum, approximately 1% of the H2AX becomes γ-phosphorylated per gray of ionizing radiation, a finding that indicates that 35 DNA double-stranded breaks, the number introduced by each gray into the 6 × 109 base pairs of a mammalian G1 genome, leads to the γ-phosphorylation of H2AX distributed over 1% of the chromatin. Thus, about 0.03% of the chromatin appears to be involved per DNA double-stranded break. This value, which corresponds to about 2 × 106 base pairs of DNA per double-stranded break, indicates that large amounts of chromatin are involved with each DNA double-stranded break. Thus, γ-H2AX formation is a rapid and sensitive cellular response to the presence of DNA double-stranded breaks, a response that may provide insight into higher order chromatin structures. In eucaryotes, DNA is packaged into nucleosomes, which are in turn arranged in various higher order structures to form chromatin (1Van Holde K.E. Chromatin. Springer-Verlag New York Inc., New York1989Crossref Google Scholar, 2Pruss D. Hayes J.J. Wolffe A.P. BioEssays. 1995; 17: 161-170Crossref PubMed Scopus (102) Google Scholar). The nucleosome, the crystallographic structure of which has recently been elucidated (3Luger K. Mader A.W. Richmond R.K. Sargent D.F. Richmond T.J. Nature. 1997; 389: 251-260Crossref PubMed Scopus (6991) Google Scholar), is composed of about 145 bp 1The abbreviations used are: bp, base pair(s); Gy, gray; CHO, Chinese hamster ovary; TEMED,N,N,N′,N′-tetramethylethylenediamine; BrdUrd, bromodeoxyuridine; AUT, acetic acid-urea-Triton X-100; AUC, acetic acid-urea-cetyltrimethylammonium bromide; DNA-PK, DNA-protein kinase. of DNA and eight histone proteins, two from each of four histone protein families, H4, H3, H2B, and H2A. In mammals, each histone family is encoded by multiple genes, which with few exceptions are expressed in concert with replication (4Heintz N. Biochim. Biophys. Acta. 1991; 1088: 327-339Crossref PubMed Scopus (138) Google Scholar). The various members of the H4, H3, and H2B families differ in few if any amino acid residues (5Baxevanis A.D. Landsman D. Nucleic Acids Res. 1996; 24: 245-247Crossref PubMed Scopus (36) Google Scholar). 2The HHGRI/NCBI Histone Sequence Database is available on the World Wide Web athttp://www.ncbi.nlm.nih.gov/Baxevani/HISTONES. In contrast, the H2A family includes three subfamilies whose members contain characteristic sequence elements that have been conserved independently throughout eucaryotic evolution (6Thatcher T.H. Gorovsky M.A. Nucleic Acids Res. 1994; 22: 174-179Crossref PubMed Scopus (174) Google Scholar, 7West M.H.P. Bonner W.M. Biochemistry. 1980; 19: 3238-3245Crossref PubMed Scopus (219) Google Scholar). The three H2A subfamiles are the H2A1-H2A2, the H2AZ, and the H2AX; in mammals the H2AZ represents about 10% of the H2A complement, the H2AX represents 2–25%, and the H2A1-H2A2 represents the balance. In addition, histone species are often modified with phosphate and acetate moieties on specific serine and lysine residues, respectively, usually near the amino or carboxyl termini. A specific role for histone acetylation has been confirmed with the finding that histone acetylases are transcription factors (8Brownell J.E. Zhou J. Ranalli T. Kobayashi R. Edmondson D.G. Roth S.Y. Allis C.D. Cell. 1996; 84: 843-851Abstract Full Text Full Text PDF PubMed Scopus (1292) Google Scholar). Consistent with this is the finding that H4 is acetylated to higher levels in euchromatin than in heterochromatin (9O'Neill L.P. Turner B.M. EMBO J. 1995; 14: 3946-3957Crossref PubMed Scopus (234) Google Scholar). Several histone modifications are correlated with chromosome condensation and mitosis; histone H3 becomes phosphorylated on residue serine 10 (10Gurley L.R. D'Anna J.A. Barham S.S. Deaven L.L. Tobey R.A. Eur. J. Biochem. 1978; 84: 1-15Crossref PubMed Scopus (407) Google Scholar), and linker histone H1 becomes multiply phosphorylated (11Bradbury E.M. Inglis R.J. Matthews H.R. Nature. 1974; 247: 257-261Crossref PubMed Scopus (285) Google Scholar). In this report, we demonstrate that H2AX becomes phosphorylated on residue serine 139 in cells when double-stranded breaks are introduced into the DNA by ionizing radiation. One of the three H2A subfamilies that has been conserved throughout evolution (12Mannironi C. Bonner W.M. Hatch C.L. Nucleic Acids Res. 1989; 17: 9113-9126Crossref PubMed Scopus (127) Google Scholar), H2AX comprises 2–10% of the H2A complement in mammalian tissues and larger fractions in lower eucaryotes where in budding yeast H2AX constitutes virtually all of the H2A (5Baxevanis A.D. Landsman D. Nucleic Acids Res. 1996; 24: 245-247Crossref PubMed Scopus (36) Google Scholar). Our finding of a human astrocytoma cell line SF268 in which H2AX is 25% of the H2A complement shows that H2AX can be more than 10% of H2A complement in tissue culture cells. The sequence that differentiates the H2AX from 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 γ-phosphorylation. This report is the first demonstration of a unique in vivo function for H2AX, a function that clearly differentiates it from the other H2A species. We report that exposure of cell cultures and mice to survivable as well as lethal amounts of ionizing radiation leads to the induction of γ-H2AX. Ionizing radiation has been present during the evolution of living systems; current background levels, about 0.5 millisieverts/year, induce on the order of 105 DNA double-stranded breaks each second in the cells of a 50-kg mammal. In tissue culture, of every 40 DNA double-stranded breaks introduced per cell by ionizing radiation, approximately one major karyotypic defect is found (13Ward J.F. Glass W.A. Varma M.N. DNA Damage and Repair in Physical and Chemical Mechanisms in Molecular Radiation Biology. Plenum Publishing Corp., New York1991Google Scholar), defects that may reflect an unbalanced genome and altered cellular metabolism, perhaps leading to cell death or neoplastic progression. We demonstrate that γ-H2AX formation is both a rapid and sensitive response to ionizing radiation. Half-maximal amounts of γ-H2AX are reached by 1 min postirradiation, and maximal amounts are reached by 10 min. At the maximum, approximately 1% of the H2AX becomes γ-phosphorylated per Gy of ionizing radiation. This value, which corresponds to about 2 × 106 bp of DNA/double-stranded break, indicates that substantial amounts of chromatin may be involved with each DNA double-stranded break. Thus, γ-H2AX formation is a rapid and sensitive cellular response to the presence of DNA double-stranded breaks, a response that may provide insight into higher order chromatin structures. The cell cultures used in this study were grown in 10-cm dishes with RPMI 1640 medium containing 10% fetal calf serum. Nuclei from approximately 107 cells were isolated essentially as described by Whitlock et al.(14Whitlock Jr., J.P. Galeazzi D. Schulman H. J. Biol. Chem. 1983; 258: 1299-1304Abstract Full Text PDF PubMed Google Scholar). Cell monolayers were washed with cold phosphate-buffered saline. One ml of lysis buffer (10 mm Tris-HCl, pH 8, 5 mm MgCl2, 0.5% Nonidet P-40) was added to each of the cell layers, which were scraped into microcentrifuge tubes, and the nuclei were pelleted for 2 s in a microcentrifuge. The histones were extracted from the pellets with 3 volumes of 0.5m HCl for 30 min on ice and prepared for two-dimensional gel analysis. For labeling studies, nuclear pellets were resuspended in 1 volume of TMCD assay buffer (10 mm Tris-HCl, pH 8, 5 mmMgCl2, 5 mm CaCl2, 5 mmdithiothreitol). One μl of [γ-32PO4]ATP was added to 9 μl of each of the nuclear suspensions, and the mixtures were incubated at 20 °C for 20 min. Then 100 μl of ice-cold assay buffer was added to each of the reaction mixtures, which were spun at 1000 rpm in a microcentrifuge for 5 min; the histones were extracted from the pellets with 3 volumes of 0.5 m HCl for 30 min on ice and prepared for two-dimensional gel analysis. The medium of CHO, SF268 or other cell cultures was replaced with 10 ml of ice-cold medium, and the cultures were exposed to a 137Cs source at a rate of either 5 or 17 Gy/min in a Shepherd Mark I irradiator. The temperature of the medium remained below 8 °C during the irradiation. After irradiation, the cold medium was replaced with medium at 37 °C, and the cultures were returned to the incubator for the times indicated. The nuclei and histones were then prepared for analysis. DBA/2 mice, 35 days old, were irradiated with 200 Gy for 12.5 min at 17 Gy/min or with 3.6 Gy for 1.5 min at 2.4 Gy/min. The mice were euthanized in a CO2 chamber at the appropriate times. The livers, about 0.75 g, wet weight, were removed, diced, and homogenized (Polytron, Brinkmann Instruments) in 5 ml of an ice-cold buffer (10 mm Tris-HCl, pH 7.5, 1 mm MgCl2) for 10 s. Nonidet P-40 was added to a final concentration of 0.5%, and the suspension was homogenized another 10 s at a slow speed to minimize foaming. The nuclei were pelleted from a 2-ml aliquot of each homogenate by a 2-s spin in a microcentrifuge. Concentrated HCl was added to the pellets to a final concentration of 0.5 mHCl; histones were extracted for analysis. Nuclear suspensions were pelleted for 2 s in a microcentrifuge; the pellets were resuspended in 0.5 m HCl and extracted for 30 min on ice. Reaction mixtures containing unbound histone were made 0.5m in HCl and extracted as above. Acid-insoluble material was pelleted for 5 min in the microcentrifuge, and the supernatants were removed to other tubes. Powdered urea was added to each of the supernatants to 8 m, phenolphthalein was added to 0.002%, and concentrated ammonia was added until the solutions became pink. Acetic acid was then added to 1 m, and the samples were loaded onto polyacrylamide gels. Histone gels comprise a first acetic acid-urea-Triton X-100 (AUT) dimension followed by a second acetic acid-urea-cetyltrimethylammonium bromide (AUC) dimension (15Bonner W.M. West M.H.P. Stedman J.D. Eur. J. Biochem. 1980; 109: 17-23Crossref PubMed Scopus (194) Google Scholar). AUT gels were prepared in shells 36 cm wide, 45 cm high, and 0.4 mm thick. The resolving gel solution contained urea (8 m), acrylamide (12%), bisacrylamide (0.11%), acetic acid (1 m), ammonia (0.03 m), Triton X-100 (0.5%), TEMED (0.5%), and riboflavin (0.0004%). The solution was degassed, poured into the shells (leaving 4 cm at the top), overlayered with water-saturated butanol, and polymerized between two fluorescent light boxes for 30 min. The stacking gel solution contained urea (8 m), acrylamide (5%), bisacrylamide (0.16%), acetic acid (1 m), ammonia (0.03 m), TEMED (0.5%), and riboflavin (0.0004%). When the resolving gels had polymerized, the butanol was removed. The stacking gel solution was degassed and 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 reservoir buffer contained acetic acid (1 m), and glycine (0.1 m). After the samples were loaded, electrophoresis was performed at 10 watts overnight. Finished gels were stained in a solution containing acetic acid (5%), ethanol (40%), and Coomassie Brilliant Blue R-250 (0.4%) for 30 min and destained for 30 min in a solution containing acetic acid (5%) and ethanol (20%). 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 (18.5%), bisacrylamide (0.11%), acetic acid (1m), ammonia (0.03 m), TEMED (0.5%), and riboflavin (0.0004%). The solution was degassed, poured into the shells (leaving 4 cm at the top), overlayered with water-saturated butanol, and polymerized between two fluorescent light boxes for 30 min. The stacking gel solution contained urea (5 m), acrylamide (5%), bisacrylamide (0.16%), acetic acid (1m), ammonia (0.03 m), TEMED (0.5%), and riboflavin (0.0004%). When the resolving gels had polymerized, the butanol was removed. The stacking gel solution was degassed, poured into the shells (leaving 2 cm at the top), and polymerized between two fluorescent light boxes for 30 min. Regions of interest were excised from the stained first dimension gels and incubated in a solution containing acetic acid (1 m), ammonia (0.03 m), and mercaptoethylamine (1%) for 30 min. The pieces were slid into the top of a second dimension gel until they rested on the stacking gel. A solution containing 1% melted agarose, acetic acid (1 m), and ammonia (0.03 m) was poured around and to the top of the inserted sample gel; the agarose was allowed to solidify. The reservoir buffer contained acetic acid (1m), glycine (0.1 m), and CTAB (0.15%; Sigma H-9151; hexadecyltrimethylammonium bromide). Electrophoresis was started at 67 milliamps/gel. This was about 12 watts/gel; when the wattage reached 26 watts/gel, the setting was switched to constant wattage at 26 watts until the Coomassie Blue migrated to the bottom of the gel. The total time of electrophoresis was about 7 h. Finished gels were stained in a solution containing acetic acid (5%), ethanol (40%), and Coomassie Brilliant Blue R-250 (0.4%) for 2 h and destained in a solution containing acetic acid (5%) and ethanol (20%). These gel recipes were used for all of the AUT-AUC gels presented in this paper except those shown in Fig. 1, which contained 18.5% acrylamide in the first AUT dimension; this higher concentration permitted the separation of all of the histone species but with some loss of resolution in the H2A region (Fig. 1 A versus Fig. 4 A). The Coomassie Blue-stained gels were recorded as TIFF images with the Eagleeye II (Stratagene Cloning Systems), the relevant images were assembled with Paint Shop Pro (Jasc, Inc) and Powerpoint (Microsoft), and the figures were printed with an HP OfficeJet Pro 1150C printer (Hewlett Packard).Figure 4Phosphorylation of γ-components: AUT-AUC gels. SF268 cells were grown almost to confluence on 10-cm dishes.A and C, one dish was incubated for 30 min at 37 °C with 5 ml of PO4-free RPMI 1640 medium with 10% fetal calf serum and containing 1 mCi of 32PO4(1000 mCi/mmol; NEN Life Science Products). B and D, a duplicate dish received 50 Gy on ice and then was incubated as above. Histones were extracted and analyzed as described under "Experimental Procedures." A and B, Coomassie Blue stain. C and D, autoradiograph. The position of the main novel component is noted as γ with anarrow when it is present and with a dotted linewhen it is absent or present in a very low amount. The dotted boxes outline the ubiquitinated H2A region in panels A–D, and in panels C and D, a longer exposure of the boxed area is reproduced in the upper right corner. The other nomenclature is explained in the legends to Figs. 1 and 3.View Large Image Figure ViewerDownload Hi-res image Download (PPT) PCR was performed on plasmids containing the coding sequences for human H2A1, H2AZ, and H2AX (12Mannironi C. Bonner W.M. Hatch C.L. Nucleic Acids Res. 1989; 17: 9113-9126Crossref PubMed Scopus (127) Google Scholar), maintaining the ATG codon at the 5′-end of the coding sequence, adding a HindIII site just upstream of the ATG codon and a convenient restriction site at the 3′-end so that the PCR fragments could be cloned in phase into the HindIII site of the pET17xb vector (Novagen, Inc.). This procedure permitted the histone species to be expressed as part of fusion proteins. After constructs were checked by sequencing, duplex oligonucleotides coding for the formic acid-sensitive sequence (Asp-Pro)6 followed by the nickel-binding sequence His6 were inserted in phase at the HindIII site. The constructs were expressed in bacterial strain BL21(DE3)pLysS (Novagen, Inc.). When expression was maximal, the bacteria were harvested; the pellets were dissolved in 3 volumes of 98% formic acid and incubated at 37 °C overnight, leading to cleavage of the fusion protein species in the (Asp-Pro)6region. The formic acid was neutralized with ammonia; the solutions were dialyzed versus 10 mm Tris-HCl, pH 7.6, overnight and passed over a nickel column in the appropriate buffer (Novagen, Inc.). The histone species with their His6 tags were eluted with an imidazole gradient. The eluted material was treated with CNOBr to cleave the tagged histone species at the methionine residue of the initiation codon, lyophilized, dissolved in the appropriate buffer, and passed through a nickel column to remove the His6-containing oligopeptides. The histone species were collected in the flow-through and stored at −20 °C. The recombinant histone species can be reconstituted in vitro into nucleosomes. 3V. S. Ivanova and W. M. Bonner, unpublished observations. Histone H2AX mutant constructs were prepared by inserting appropriate duplex oligonucleotides at a SfiI site, unique in the H2AX-pET17xb expression vector and situated in the codon for residue threonine 136. HeLa nuclear extracts were prepared from resuspended nuclear pellets by adding 0.1 volume of 5 m NaCl to the latter. The residual nuclei were pelleted, and the kinase extracts were used immediately. Reaction mixes (10 μl) contained 1 μl of 10 × TMCD assay buffer; 1 μg of recombinant H2A1, H2AX, or mutant H2AX construct; 1 μl of [γ-32PO4]ATP; and 1 μl of nuclear kinase extract in the appropriate assay buffer. After incubation for 20 min at 20 °C, the reactions were terminated, and the histone proteins were analyzed either by two-dimensional AUT-AUC or by one-dimensional SDS gel electrophoresis. When mammalian cell cultures are exposed to ionizing radiation and the acid-soluble nuclear proteins are analyzed on two-dimensional AUT-AUC gels, novel components that will be referred to as γ (Fig. 1, A and B) are found in the H2A region of these gels. In the first AUT dimension, histones separate according to peptide length, charge, and the ability to partition onto Triton X-100 micelles. The ability to bind Triton X-100 micelles is a property of all the known core histone species. Sensitive to single amino acid differences (16Zweidler A. Methods Cell Biol. 1978; 17: 223-233Crossref PubMed Scopus (313) Google Scholar), this property enables closely related histone species to be resolved. Since the micelles are uncharged, protein molecules partitioning onto Triton X-100 micelles are retarded; this partitioning and hence the retardation can be modulated by the concentration of urea in the gel. In the second AUC dimension, the histones separate according to peptide length, charge, and shape. This combination of separation parameters resolves the histones from all other proteins on these two-dimensional gels. In addition to permitting the resolution of closely related histone gene products, these gels also permit the separation of post-translationally modified forms of the histone species (15Bonner W.M. West M.H.P. Stedman J.D. Eur. J. Biochem. 1980; 109: 17-23Crossref PubMed Scopus (194) Google Scholar). These species, which differ by single charges from each other, generally migrate just behind the parent species in both dimensions, thus forming a diagonal line (most apparent for H4 in Fig. 1, A and B). The charge differences arise most often from the phosphorylation of serine residues, which adds one negative charge to the protein and from the acetylation of lysine residues, which removes one positive charge from the protein (17Pantazis P. Bonner W.M. J. Biol. Chem. 1981; 256: 4669-4675Abstract Full Text PDF PubMed Google Scholar). H2A (18Goldknopf I.L. Rosenbaum F. Steiner R. Vidali G. Allfrey V.G. Busch H. Biochem. Biophys. Res. Commun. 1979; 90: 269-277Crossref PubMed Scopus (14) Google Scholar) and to a lesser extent H2B (19West M.H.P. Bonner W.M. Nucleic Acids Res. 1981; 20: 4671-4680Google Scholar) also have ubiquitin adducts; because of the size of ubiquitin, these adducts migrate in a separate region of the gel (Fig. 1, A and B). AUT-AUC gel analysis has been performed on other mammalian cell lines after 137Cs irradiation, including normal human fibroblast IMR90, transformed human fibroblast VA13, hamster CHO, human HeLa, and human HL60; all yielded similar results. Thus, γ-components are induced by ionizing radiation in a wide variety of mammalian cells. To help elucidate the physiological relevance of γ-components, we examined whether or not they are inducible under survivable conditions. SF268 cultures were exposed to 1.2, 3.6, or 10.8 Gy of ionizing radiation and permitted to recover for 30 min. γ-Components were apparent in all three cases (Fig. 2,B–D). While irradiated cells are metabolically active for several days, they may not be able to reproduce. However, cloning analysis of duplicate SF268 cell cultures showed that there was at least 40% clonal survival at 1.2 Gy and 10% clonal survival at 3.6 Gy (data not shown). Thus, γ-components form in cell cultures under survivable conditions. To determine whether or not the induction of γ-components is a response seen in whole organisms, we exposed mice to ionizing radiation and extracted the histones from their livers. Mice were exposed to 3.6 Gy, which is 60% of the 6-Gy 30LD50 (50% mortality 30 days after exposure) (20Ueda T. Toyoshima Y. Moritani T. Ri K. Otsuki N. Kushihashi T. Yasuhara H. Hishida T. Int. J. Radiat. Biol. 1996; 69: 199-204Crossref PubMed Scopus (27) Google Scholar, 21Hornsey S. Hevesy G.C. Forssberg A.G. Abbatt J.D. Advances in Radiobiology. Charles C. Thomas, Springfield, IL1957: 248Google Scholar, 22Thomson J.F. Radiation Protection in Animals. Reinhold, New York1962Google Scholar); these mice would be expected on average to have a life span shortened by only 10–15% (23Casarett A.P. Radiation Biology. Prentice Hall, Englewood Cliffs, NJ1968Google Scholar). γ-Components were apparent 15 (Fig. 2 F) and 40 min (Fig. 2 G) after exposure to 3.6 Gy. γ-Components were more abundant when mice were exposed to 200 Gy (Fig. 2 H), which kills mice within several hours. Thus, γ-components form in living organisms at both nonlethal and lethal amounts of ionizing radiation. Since the formation of γ-components appeared to be a widespread cellular reaction to ionizing radiation among mammals, it is relevant to determine whether the cell cultures are responding directly to the ionizing radiation or to a particular type of cellular damage induced by the ionizing radiation. Ionizing radiation introduces many different kinds of damage into cells, directly by collision with atoms of biological molecules and indirectly by collision with water molecules. The latter generates free radicals, of which the most abundant is the hydroxyl radical. Ionizing radiation produces high local concentrations of hydroxyl radicals that, if located next to a DNA molecule, may produce locally multiply damaged sites (13Ward J.F. Glass W.A. Varma M.N. DNA Damage and Repair in Physical and Chemical Mechanisms in Molecular Radiation Biology. Plenum Publishing Corp., New York1991Google Scholar) containing alterations of the base and sugar residues and breaks of one or both strands of the DNA double helix. Several agents and procedures that do or do not introduce double-stranded breaks into the DNA in cells were examined (Fig. 3). Cellular DNA can be sensitized to form DNA double- and single-stranded breaks upon irradiation with ultraviolet A light (350 nm) when cell cultures are grown in the presence of BrdUrd and incubated with Hoechst dye 33258 just before irradiation (24Limoli C.L. Ward J.F. Radiation Res. 1993; 134: 160-169Crossref PubMed Scopus (111) Google Scholar). Like ionizing radiation, this method introduces double-stranded breaks as well as single-stranded breaks into DNA, but unlike the former, the mechanism is nonradiolytic. The procedure was found to result in the formation of γ-components in SF268 cells (Fig. 3, A–D) but only if BrdUrd, dye, and light were all present. Since this procedure leads to the formation of DNA breaks by a nonradiolytic mechanism and without hydroxyl radical formation in cells, γ-components are not a cellular response directly to ionizing radiation or to the presence of hydroxyl radicals, but to the presence of DNA breaks. This result was substantiated by the presence of γ-components when SF268 cell cultures were incubated with bleomycin (25Chabner B. Longo D.L. Cancer Chemotherapy and Biotherapy: Principles and Practice. 2nd Ed. Lippincott-Raven Publishers, Philadelphia1996: 379-393Google Scholar), a compound that also introduces double- and single-stranded breaks into cellular DNA by a nonradiolytic mechanism (Fig. 3 E). While the above described procedures introduce DNA double- and single-stranded breaks without hydroxyl radical formation, H2O2 produces hydroxyl radicals and DNA single-stranded breaks, as does ionizing radiation, but does not produce significant amounts of DNA double-stranded breaks because the cellular distribution of the hydroxyl radicals differs between the two agents (26Friedberg E.C. DNA Repair and Mutagenesis. American Society for Microbiology Press, Washington, D. C.1995: 1-58Google Scholar). With H2O2, radicals are generated homogeneously throughout the cell as contrasted to the heterogeneous distribution found with ionizing radiation. Incubation of SF268 cell cultures with 10 μm (Fig. 3 F) or 50 μm (Fig. 3 G) H2O2 for 30 min did not lead to detectable formation of γ-components, although these cultures were still able to form γ-components after exposure to ionizing radiation (Fig. 3 H). These concentrations are damaging to cells; incubation of CHO cultures with 50 μmH2O2 for 30 min at 37 °C was found to result in approximately 95% clonal lethality (27Sestili P. Cantoni O. Cattabeni F. Murray D. Biochim. Biophys. Acta. 1995; 31: 130-136Crossref Scopus (20) Google Scholar). Another agent that damages cellular DNA primarily by introducing single-strand lesions (26Friedberg E.C. DNA Repair and Mutagenesis. American Society for Microbiology Press, Washington, D. C.1995: 1-58Google Scholar) is ultraviolet C light. When SF268 cell cultures were irradiated with 1, 3, 10, 30, or 100 J/m2 of ultraviolet C radiation, amounts of radiation that cover the range from little if any cellular effect to complete lethality, no γ-components were detected after a 30-min recovery (data not shown). Thus, it is the DNA double-stranded break from ionizing radiation that is responsible for γ-component formation. The AUT-AUC gels shown in Fig. 1 were prepared with a 18% first dimension AUT gel to resolve all histone species. The AUT-AUC gels shown in the other figures were prepared with a 12% first dimension AUT gel to optimize the separation of γ-components; however, H4, H2B, and several of the H3 isoforms migrate at the buffer front in this dimension and thus are separated only in the second. γ-Components were obtained when SF268 cultures were exposed to 50 Gy of ionizing radiation and returned to a 37 °C incubator for a 30-min recovery period (Figs. 1 B and 4 B). When 32PO4 was included in the medium during the recovery period, γ-components became radioactively labeled (Fig. 4 D). The pattern of the