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 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
, these components are formed in
vivo by several procedures that introduce double-stranded breaks
into DNA.
-Components, which appeared to be the only major novel
components detected by mass or 32PO4
incorporation 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.
-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
-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
-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,
-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.
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INTRODUCTION |
In eucaryotes, DNA is packaged into nucleosomes, which are in turn
arranged in various higher order structures to form chromatin (1, 2).
The nucleosome, the crystallographic structure of which has recently
been elucidated (3), is composed of about 145 bp1 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
(4). The various members of the H4, H3, and H2B families differ in few
if any amino acid residues
(5).2 In contrast, the H2A
family includes three subfamilies whose members contain characteristic
sequence elements that have been conserved independently throughout
eucaryotic evolution (6, 7). 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 (8). Consistent with this is the finding that
H4 is acetylated to higher levels in euchromatin than in
heterochromatin (9). Several histone modifications are correlated with
chromosome condensation and mitosis; histone H3 becomes phosphorylated
on residue serine 10 (10), and linker histone H1 becomes multiply
phosphorylated (11).
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 (12), 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 (5). 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
-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
-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 (13), defects that may reflect an unbalanced genome and
altered cellular metabolism, perhaps leading to cell death or
neoplastic progression.
We demonstrate that
-H2AX formation is both a rapid and sensitive
response to ionizing radiation. Half-maximal amounts of
-H2AX are
reached by 1 min postirradiation, and maximal amounts are reached by 10 min. At the maximum, approximately 1% of the H2AX becomes
-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,
-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.
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EXPERIMENTAL PROCEDURES |
Isolation and Labeling of Nuclei--
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. (14). 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 0.5 M 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 mM
MgCl2, 5 mM CaCl2, 5 mM
dithiothreitol). One µl of [
-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.
Exposure of Cell Cultures to Ionizing Radiation--
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.
Exposure of Mice to Ionizing Radiation--
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 M
HCl; histones were extracted for analysis.
Two-dimensional Gel Analysis of Histones--
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 0.5 M 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 (15). 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 (1 M), 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 (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, 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 (1 M), 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. 1A versus Fig. 4A). 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).
Preparation of Recombinant H2AX--
PCR was performed on
plasmids containing the coding sequences for human H2A1, H2AZ, and H2AX
(12), 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)6 region. 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.3 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.
Nuclear Extract Labeling of Recombinant H2AX--
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
[
-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.
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RESULTS |
Ionizing Radiation Induces Novel Protein Components Resolvable on
Histone Gels--
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
(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 (16), 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.

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Fig. 1.
Formation of novel components in cells after
ionizing radiation. SF268 cell cultures were exposed to 50 Gy from
a 137Cs source at the rate of 17 Gy/min and returned to the
37 °C incubator for 30 min. Histones were extracted and analyzed as
described under "Experimental Procedures." The panels
present gels containing 18.5% acrylamide in the AUT first dimension,
while the insets present the H2A portion of gels containing
12% acrylamide in the AUT first dimension. A, control.
B, gel exposed to 50 Gy. Histone species are noted;
uH2A refers to ubiquitinated H2A species. The main novel
component is noted as .
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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 (15). 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 (17). H2A (18) and to a lesser extent H2B (19) 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).
-Components Are Formed in Cell Cultures and Mice under Nonlethal
Conditions--
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,
-components are induced by ionizing radiation in a wide
variety of mammalian cells.
To help elucidate the physiological relevance of
-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.
-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,
-components form in cell cultures under
survivable conditions.

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Fig. 2.
Induction of -components in cells and mice
by nonlethal 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 analyzed
as described under "Experimental Results." E-H, mice were irradiated, and their liver histones were prepared and analyzed as
described under "Experimental Procedures." E,
unirradiated mouse. F, mouse irradiated with 3.6 Gy over 1.5 min and sacrificed 15 min afterward. G, mouse irradiated
with 3.6 Gy over 1.5 min and sacrificed 40 min afterward. H,
mouse irradiated with 200 Gy over 12 min and sacrificed 18 min later.
The position of the main novel component is noted as with an
arrow when it is present and with a dotted line
when it is absent or present in very low amount. In the case of mouse
liver, a second arrow denotes a another -component that
migrates faster in the second dimension.
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To determine whether or not the induction of
-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) (20-22); these mice would be
expected on average to have a life span shortened by only 10-15%
(23).
-Components were apparent 15 (Fig. 2F) and 40 min
(Fig. 2G) after exposure to 3.6 Gy.
-Components were more abundant when mice were exposed to 200 Gy (Fig. 2H), which
kills mice within several hours. Thus,
-components form in living
organisms at both nonlethal and lethal amounts of ionizing
radiation.
-Components Are Induced by DNA Double-stranded
Breaks--
Since the formation of
-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 (13)
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 (24). 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
-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,
-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
-components when SF268 cell cultures were incubated with
bleomycin (25), a compound that also introduces double- and
single-stranded breaks into cellular DNA by a nonradiolytic mechanism
(Fig. 3E).

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Fig. 3.
Formation of -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 µM BrdUrd
and 2.5 µM 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 µg/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/m2 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 µM
(F) or 50 µM (G and H)
H2O2 for 30 min at 37 °C. The
H2O2-containing medium was 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 with an arrow when it is present and with a
dotted line when it is absent or present in a very low
amount. H2AX S1/K5 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 -component that migrates faster in the second
dimension.
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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 (26). 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. 3F) or 50 µM (Fig. 3G) H2O2 for
30 min did not lead to detectable formation of
-components, although
these cultures were still able to form
-components after exposure to
ionizing radiation (Fig. 3H). These concentrations are
damaging to cells; incubation of CHO cultures with 50 µM
H2O2 for 30 min at 37 °C was found to result
in approximately 95% clonal lethality (27). Another agent that damages
cellular DNA primarily by introducing single-strand lesions (26) 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
-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
-component
formation.
-Components Are Phosphorylated H2A Derivatives--
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
-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.
-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. 1B
and 4B). When 32PO4 was included in
the medium during the recovery period,
-components became
radioactively labeled (Fig.
4D). The pattern of the
-components appeared to mimic the pattern of the H2AX species with
its previously characterized modified forms (17). H2AX, as well as H2A1
and H2A2, can be phosphorylated on residue serine 1 and/or acetylated on residue lysine 5 (noted as H2A
S1/K5 on Fig. 4A) and/or
ubiquitinated on residue lysine 119 (uH2A, inside the dotted
rectangle in Fig. 4A). The phosphorylation of serine 1 accounts for the presence of 32PO4 label found
in the H2A region in the control cultures (noted as H2A
S1 in Fig. 4B). After 137Cs
irradiation, labeling on H2A serine 1 was decreased, while the
-components became heavily labeled (Fig. 4, C and
D). Note that the
-components appear to be ubiquitinated
to the same extent as the H2A species when detected by mass (inside the
dotted rectangles, Fig. 4, A and B)
and by label (inside the dotted rectangles, Fig. 4,
C and D). These results from AUT-AUC gels
indicate that the
-components are phosphorylated H2A
derivatives.

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Fig. 4.
Phosphorylation of -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 with an
arrow when it is present and with a dotted line
when 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.
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To obtain more information about the identity of
-components,
similarly prepared samples were subjected to SDS-AUC gel analysis. In
SDS gels (Fig. 5A), proteins
separate primarily according to size. H2AX migrates almost
coincidentally with H3 in SDS gels, but the two are resolved on AUC
gels (Fig. 5, B and C).
-Components were not
apparent on SDS-AUC gels by Coomassie Blue stain (Fig. 5, B
and C) but were apparent when the
32PO4 label was detected (Fig. 5, D
and E), because
-components migrate coincidentally with
H3 in both SDS and AUC gels, but not in AUT gels. Thus, to separate
-components from other proteins it is necessary to utilize AUT gels
to resolve the histone species followed by AUC gels to resolve the
-components from the known modified forms of H2AX.

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Fig. 5.
Phosphorylation of -components: SDS-AUC
gels. SF268 cells were grown almost to confluence on 10-cm dishes.
A, first dimension SDS gel used for the two-dimensional gel
presented in panel B. B and D, one
culture was incubated for 30 min at 37 °C in 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). C and E, a duplicate culture 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 with an arrow when it is
present and with a dotted line when it is absent or present
in a very low amount. The ubiquitinated H2A region shown in
panels D and E is from a longer exposure. The
other nomenclature is explained in the legends to Figs. 1 and 3.
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AUT-AUC and SDS-AUC gels contain many other proteins, only some of
which are detectable by Coomassie Blue stain. Of components that were
visible in these experiments either by stain or by
32PO4 labeling, the
-components were by far
the most heavily labeled and the only major ones induced by
137Cs irradiation (Figs. 4 and 5). It is also apparent that
either with or without 137Cs irradiation of these cultures,
there was no significant 32PO4 incorporation
into H3 (Fig. 4, C and D) or into the H2B and H4
species (Fig. 5, D and E).
-Components Can Be Formed in Vitro--
We had found that
32PO4-labeled
-components can be generated
in vitro with isolated nuclei and in nuclear extracts with
recombinant H2AX. These findings stemmed from reports that a histone,
originally identified as H3 from SDS gels (14, 28, 29) and later as H2AX (30), was the only protein labeled to a significant extent when
isolated nuclei were incubated with
[
-32PO4]ATP. The report of H2AX as the
labeled histone species (30) utilized an AUT-SDS gel system and thus
did not resolve
-components from H2AX, since, as shown in Figs. 4
and 5,
-components and H2AX do not resolve from each other in either
of these systems. However, with the multiplicity of
-components
(Fig. 6B), the relationship of
-components to H2AX appears even more compelling. More components
are visible for two reasons. The first is the higher specific activity
attainable with in vitro radioactive labeling. The second is
the growth of the cells in 5 mM sodium butyrate for several
hours before harvest, a condition that leads to increased acetylation
of histone (14). Under these in vitro labeling conditions,
mass amounts of
-components are not expected, since there is no
source of bulk phosphate 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 introduction of
DNA double-stranded breaks during the centrifugation steps used to
isolate nuclei.

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Fig. 6.
-component formation in vitro.
A and B, nuclei prepared from SF268 cells were
incubated with [ -32PO4]ATP as described
under "Experimental Procedures." The cell cultures were incubated
for 3 h before nuclear isolation with 5 mM sodium
butyrate to increase in vitro labeling (14). C
and D, phosphorylation of recombinant H2AX by nuclear
extract as described under "Experimental Procedures." Carrier
histone from SF268 cells irradiated with 50 Gy was added before loading
the gel. A and C, Coomassie Blue stain.
B and D, autoradiographs. The position of the
main novel component is noted as with an arrow when it is present and with a dotted line when it is absent or
present in a very low amount. H2AX
S1&K5 refers to H2AX species 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.
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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 species and examined their
ability to be phosphorylated with 0.35 M NaCl extracts. Only H2AX could be labeled with
[
-32PO4]ATP to a significant extent
with these extracts; in addition, the labeled material migrated
primarily as
-components (Fig. 6, C and D).
-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 phosphorylated on residue serine 1 (data not
shown). Thus, these in vitro experiments confirm that
-components are H2AX derivatives.
-Component Is H2AX-phosphorylated on Serine Residue
139--
The finding that recombinant H2AX forms
32PO4-labeled
-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
-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
-phosphorylation, since it has been
conserved throughout evolution in at least one H2A species in each
animal species examined.

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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 modification sites discussed in this
work are shown with the affected amino acid residue. The bacterial
expression plasmid encoding 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
techniques, and recombinant proteins were produced as described under
"Experimental Procedures."
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H2AX protein has glutamic acid residues at positions 92 and 141 with no
intervening aspartic acid and arginine residues. Digestion with the
protease StaphV8, which cleaves at aspartic and glutamic acid residues,
would be expected to yield a polypeptide 49 residues long, while
clostripain digestion would be expected to generate a 57-residue
C-terminal fragment that subsumes the 49-residue StaphV8 polypeptide
(Fig. 7). In contrast, trypsin digestion would be expected to generate
small C-terminal fragments. This C-terminal region of the H2AX sequence
is the only region in any core histone that yields similarly sized
large peptides with clostripain and StaphV8.
To confirm that
-components contain phosphate in the predicted
region, natural 32PO4-labeled
-component was
digested with clostripain, StaphV8, and trypsin. Digestion with the
first two resulted in large similarly sized radioactive fragments,
while trypsin digestion resulted in a small one (Fig.
8A). Next, recombinant H2AX
labeled with [
-32PO4]ATP and nuclear
kinase was digested with the same enzymes and was found to yield the
same pattern of fragments (Fig. 8B, the lanes noted as
0 under none (N), trypsin (T),
clostripain (C), and StaphV8 (S)) to that
obtained with the natural
-component. Thus,
-component is a form
of H2AX phosphorylated in the C-terminal region.

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Fig. 8.
Peptides of -H2AX. A, natural
-component labeled with [ -32PO4]ATP in
whole nuclei was isolated from the gel, electroeluted, dialyzed against
water, and freeze-dried. The material was dissolved in the appropriate
buffers and incubated with water (U), trypsin (T), clostripain (C), or staphylococcus V8
(S) (all from Promega). B, recombinant protein
constructs H2AX-wt, -c22, and -c23 were labeled as described under
"Experimental Procedures" and incubated as above and with
N-bromosuccinimide (Sigma). All digests were mixed with one
volume of 2 × SDS sample buffer, boiled for 5 min, and analyzed
by electrophoresis on 30% acrylamide gels containing SDS. The wet gels
were immediately exposed to film at room temperature.
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To determine which residue is involved in the
-phosphorylation of
H2AX, we prepared two recombinant H2AX derivatives with altered amino
acid sequences (Fig. 7) that would provide new cleavage sites at
predicted positions for the enzymes mentioned above. Clostripain
digestion of the parent H2AX-c0 construct labeled with nuclear extract
and [
-32PO4]ATP yielded a large but less
than full-length labeled peptide consistent with a size of 57 residues
(Fig. 7), while in construct H2AX-c22 (Fig. 7) almost all of the label
was found in a small peptide consistent with the predicted size of 8 residues (Fig. 8B). Digestion with StaphV8 also yielded new
small peptides with construct H2AX-c22 as predicted, substantiating the
result with clostripain. StaphV8 digestion would also be expected to
remove the terminal tyrosine, indicating that the phosphorylation takes place on the peptide ATQASQE (Fig. 8B). In construct
H2AX-c23, an alanine residue replaces the threonine residue to yield a
tryptic peptide AAQASQEY; that this construct is phosphorylated to a
similar extent as is H2AX-c22 indicates that threonine residue 136 is not a major phosphorylation site but may be a secondary site. Histones
do not contain tryptophan, thus N-bromosuccinimide treatment under conditions that cleave proteins only at tryptophan residues does
not cleave construct H2AX-c0 (Fig. 8B, N);
however, construct H2AX-c22 is cleaved under these conditions to yield
a small labeled peptide consistent with the expected size (Fig.
8B, N). These results demonstrate that
-components are H2AX species phosphorylated on serine 139.
Recognition Site for
-Phosphorylation--
A major advantage 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
-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 activity of the construct to 9.6% of the H2AX control, an
expected finding, since this is the site of phosphorylation; however,
glutamine 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), possibly 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 unimportant to the specificity (H2AX-c7). Changes in length
that placed serine 139 either closer to or farther from the C terminus appeared to be of lesser importance (H2AX-c2, -c3, and -c6). On the
other hand, that there may be other determinants of specificity was
indicated by the low activity of constructs H2AX-c4 and c5, in which
the SQ motif was present but in different backgrounds. These results
show that the SQ sequence is an important determinant for
-phosphorylation of H2AX.
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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.
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These findings allow us to assign structures to each of the modified
H2AX species. Fig. 9A presents
an enlargement of the H2AX and
-component region from the gel
presented in Fig. 6B. A grid is superimposed over the
pattern of H2AX and
-components; the same grid is reproduced in Fig.
9B with modification sites, as noted in the H2AX sequence
(Fig. 7), assigned to each of the nine forms. The pattern of
ubiquitinated forms is identical to that of the nonubiquitinated forms
(Fig. 6B, inset), except that each H2AX molecule
also contains a ubiquitin modification on residue lysine 119. These
assignments also provide an explanation for the different relative
amounts of a second faster
-component seen in mouse and human cells
(second arrow in Fig. 2, F-H, and Fig. 3,
A and E). Mouse H2AX contains a serine residue at
position 136 as well as at position 139 (31), while the human form has a threonine residue at position 136. If serine is the preferred substrate for the kinase, then mouse
-H2AX might be expected to
contain more of a doubly
-phosphorylated H2AX.

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Fig. 9.
Assignment of -H2AX derivatives to
particular structures. A, an enlargement of the in
vitro labeled material presented in Fig. 6B with a grid
superimposed. 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 modified in both positions.
Modifications at positions 136 and 139 are likewise noted.
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-H2AX Forms within Seconds after Exposure of Cells to Ionizing
Radiation--
Ionizing radiation can be delivered in precisely
measured amounts and time periods. In addition, its effects on cells
have been intensively investigated qualitatively and quantitatively; ionizing radiation from 137Cs results in 35 DNA
double-stranded breaks/G1 genome in mammalian cells (13,
26, 32). These parameters enable us to determine the kinetics and
stoichiometry of
-H2AX formation. To determine how quickly
-components form after exposure to ionizing radiation, hamster CHO
cell cultures were irradiated on ice with 200 Gy from a
137Cs source, rapidly returned to 37 °C, and allowed to
recover for various times.
-H2AX did not form in cell cultures on
ice but was visible 20 s after returning them to 37 °C (Fig.
10B).
-H2AX increased in
amount within 10 min to more than 50% of the total H2AX (Fig. 10,
C-F), remained at that level until about 30 min postirradiation (Fig. 10G), and then decreased over a period
of hours (Fig. 10, H-I). Densitometric analysis of the
amounts of H2AX and
-H2AX from the gels presented in Fig. 10 is
shown in Fig. 11. The data form a
smooth curve with a rapid rise and a slower decrease. While the maximum
is reached at 9-30 min, half the maximum is reached in 1 min. Although
these experiments were performed with large amounts of ionizing
radiation to obtain a significant signal at short times, similar time
courses are observed at lower amounts (data not shown). However, with
mice at nonlethal amounts of ionizing radiation, quantitation of the
images presented in Fig. 2, E-G, yielded values of
approximately 2%
-H2AX at 15 min (Fig. 2F), 5% at 40 min (Fig. 2G), and less than 2% at 70 min (data not shown),
a time course consistent with that shown in Fig. 11. Because of these
results, 30 min was chosen as the optimum recovery time when other
parameters were studied. Incubation of the irradiated cultures with the
protein synthesis inhibitor cycloheximide (100 µg/ml) did not prevent
-phosphorylation (data not shown), indicating that any proteins
necessary for
-modification are already present. Likewise,
inhibiting DNA replication with hydroxyurea (10 mM) had no
effect on
-H2AX formation.

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Fig. 10.
Relative amount of -H2AX at various times
after exposure 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 harvested immediately), and the cultures were allowed to
recover for 20 s (B), 1 min (C), 3 min
(D), 9 min (E), 15 min (F), 30 min
(G), 60 min (H) or 90 min (I).
Histones were extracted and analyzed as described under "Experimental
Procedures." TIFF images of the Coomassie Blue-stained gels of
histones H2A1 and H2AX were recorded with the Eagleeye II (Stratagene
Cloning Systems). The position of the main novel component is noted as
with an arrow when it is present and with a dotted
line when it is absent or present in a very low amount.
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Fig. 11.
Relative amount of -H2AX at various times
after exposure 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 -H2AX measured in unirradiated
cells. The open and filled symbols denote
separate experiments.
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-H2AX Formation Is Proportional to the Amount of
Radiation--
When hamster CHO cell cultures were subjected to
different amounts of ionizing radiation, the fraction of
-H2AX was
found to increase with the amount of radiation (Fig.
12, A-F). Densitometric analysis of the data is presented in Fig.
13. The initial slope of the curve for
CHO cells (filled circles) indicates that about 1.0% of the
H2AX complement became
-modified per Gy of radiation. This value may
be underestimated because it assumes that all of the relevant H2AX is
in the
-modified state simultaneously. On the other hand, the H2AX
in hamster CHO cultures receiving 200 Gy became 67%
-modified (Fig.
11F) after a 30-min recovery, indicating that the maximal
value of
-H2AX modification is probably no more than 1.5% per Gy
for CHO cells. Results with the normal human fibroblast line IMR90 were
found to be very similar to those obtained with CHO cells (data not
shown).

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Fig. 12.
The relative amount of -H2AX is
proportional to the amount of radiation: gels. Cultures of hamster
CHO cells were exposed to 0 (A), 12.5 (B), 25 (C), 50 (D), 100 (E), or 200 (F) Gy of ionizing radiation and permitted to recover at
37 °C for 30 min. Histones were extracted from the cultures and
analyzed as described. TIFF images of the Coomassie Blue-stained gels
of histones H2A1 and H2AX were recorded with the Eagleeye II
(Stratagene Cloning Systems). The position of the main novel component
is noted as with an arrow when it is present and with a
dotted line when it is absent or present in a very low
amount.
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Fig. 13.
The relative amount of -H2AX is
proportional to the amount of radiation: quantitation. Filled
circles, the H2AX components on the TIFF images presented in the
upper panel along with a duplicate set of cultures exposed
separately were quantitated with ImageQuant software version 3.3 (Molecular Dynamics) without any contrast or brightness enhancement.
Open circles and squares, quantitation of two
similar experiments performed with human SF268 cell cultures allowed to
recover for 30 min. The arrows denote the data points from
the 1.2- and 3.6-Gy samples shown in Fig. 2, B and
C.
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Densitometric results are also shown for SF268 cultures (Fig. 13,
open symbols).
-H2AX formation in this cell line appears to be more sensitive to ionizing radiation, perhaps related to its high
relative content of H2AX. Note that with SF268, the amount of
-H2AX
induced per Gy at the survivable amounts of radiation, 1.2 and 3.6 Gy
(Fig. 2, B and C; Fig. 13, arrows) is
similar to that induced at the low nonsurvivable amounts, indicating
that
-H2AX formation seen under lethal conditions is the same
process as that seen under survivable conditions. The efficiency of
-H2AX formation is similar in mice. At 40 min postirradiation (Fig. 2G), formation of
-H2AX was approximately 1.4% per Gy,
results comparable with those found in cell culture (Fig. 13).
-H2AX Modification and the Relative Abundance of
H2AX--
Previous data have shown that the fraction of H2AX converted
to
-H2AX forms is similarly dependent on the amount of ionizing radiation in both CHO cells and normal human IMR90 fibroblasts. Both
these cell lines contain H2AX as 9-10% of the total H2A complement. However, we have investigated
-H2AX formation in cell lines with H2AX comprising as little as 2.4% and as much as 25% of the total H2A
complement (Table II). Since the number
of DNA double-stranded breaks introduced per Gy per unit of chromatin
is the same irrespective of cell lineage (13), this variation enables
us to ask whether there is a constant number or a constant percentage
of
-H2AX molecules formed per DNA double-stranded break. The data in
Table II are consistent with a similar percentage but not a similar number of H2AX molecules being
-phosphorylated per DNA
double-stranded break; each Gy of ionizing radiation leads to the
-phosphorylation of about 1-2% of the H2AX irrespective of whether
the H2AX accounts for 2.5 or 25% of the total H2A complement. With the
variation in its relative abundance, H2AX is unlikely to be localized
to certain specific regions of the chromatin but is likely to be randomly distributed among the nucleosomes. Supporting this assumption is the fact that in lower eucaryotes most or all of the H2A is H2AX
(5). If H2AX is randomly distributed throughout the chromatin, then the
fraction of
-H2AX is a measure of the fraction of the chromatin and
hence of the DNA that is involved per Gy. Thus, the simplest
explanation for these findings is that a similar region of the
chromatin is involved per Gy irrespective of the cell line. One Gy of
ionizing radiation causes 35 DNA double-stranded breaks/G1
genome, which is 6 × 109 bp of DNA in mammalian
cells. If 1% of the chromatin is involved per 35 DNA double-stranded
breaks, then 0.03% is involved in each. 0.03% of 6 × 109 bp is 1.8 × 106 bp. Thus, one of the
intriguing implications of these findings is that megabase regions of
chromatin appear to be involved in each DNA double-stranded break.
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Table II
Constant percentages, not numbers, of H2AX molecules are -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 -H2AX/H2AX ratio was determined 30 min after
exposing the cell cultures to 25 Gy. The following conversion factors
and assumptions were used. 1) The mammalian G1 genome contains
6 × 109 bp of DNA, hence about 30 × 106
nucleosomes (200 bp/nucleosome) and 60 × 106 H2A
molecules (2 molecules/nucleosome). 2) 25 Gy induces about 875 DNA
double-stranded breaks per G1 genome. 3) H2AX is randomly distributed in the chromatin.
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DISCUSSION |
H2AX becomes phosphorylated on serine 139 rapidly and extensively
after exposure of mammalian cell lines and mice to various procedures
that lead to the formation of DNA double-stranded breaks.
-H2AX
formation begins within seconds after exposure to ionizing radiation
and rapidly passes through a half-maximal value at 1 min to a maximal
value at 9-30 min. DNA double-stranded breaks are repaired by various
mechanisms in mammalian cells. One pathway involves the DNA-PK complex
that is defective in scid mice (33). Evidence for a second DNA
double-stranded break repair system has recently been reported (34);
this system, which functions during G2, appears to be
normal in scid cells. H2AX is a substrate for phosphorylation by DNA-PK
in vitro (35), and we found that purified DNA-PK
-phosphorylated purified H2AX in vitro (data not shown).
However, when we examined several of the cell lines that are known to
be deficient in DNA-PK, either in the catalytic subunit or in the Ku
subunits (36, 37), neither the C.B-17-SCID mouse cell line (33), nor
the V3 hamster mutant line (38), nor the ICR-SCID mouse (39) exhibited
a noticeable deficit in
-H2AX formation after exposure to ionizing
radiation. With the above mentioned mutant cell lines and mice, it is
also possible that residual DNA-PK activity is present, since these are
not knockouts. A cell line from the Ku80 knockout mouse (40) was donated by Gloria Li. This line also showed normal
-H2AX formation. Thus,
-H2AX formation could result from another DNA double-stranded break repair system that does not utilize DNA-PKcs or from
a step upstream of DNA-PKcs action.
Human cell line M059J, a mutant line lacking DNA-PK protein (41)
donated by Joan Turner did show a substantial deficiency in
-H2AX
formation. Fifteen min after 200 Gy, the control M059K line converted
over 60% of its H2AX to
-forms, a value similar to that obtained in
other mammalian cell lines. In contrast, the mutant M059J line under
the same conditions contained no more than 25%
-H2AX. Human cells
contain about 10 times as much DNA-PK activity as do rodent cells,
indicating that there may be important differences in these enzyme
systems of the two groups.
The locus for the ataxia-telangiectasia defect is 11q23, close to that
of the H2AX. However, three ataxia-telangiectasia cell lines from
complementation groups A, C, and D were found to contain H2AX and in
addition showed no significant deficit in
-H2AX formation after
exposure to ionizing radiation (data not shown). Thus, the ataxia-telangiectasia kinase (42) is not responsible for
-H2AX formation after irradiation. The defects in other human genetic radiosensitive diseases are located on other chromosomes, suggesting that these diseases are not due to defective H2AX protein (34). H2AX
knockout cell lines and mice will help determine the role of
-H2AX
formation in cellular metabolism. We are currently investigating possible relationships between the kinase responsible for
-H2AX formation and other kinases.
At the time of maximal modification, H2AX on 1-2% of the chromatin is
-modified. Since each Gy causes 35 DNA double-stranded breaks/6 × 109 bp, this is an amount of chromatin equivalent to
1.8-3.5 × 106 bp of DNA/double-stranded break. While
this is a strikingly large amount of chromatin, the DNA double-stranded
break is a serious lesion that often leads to chromosomal
abnormalities; of the 35 DNA double-stranded breaks/genome per Gy,
approximately one will result in a visible chromosomal abnormality, and
more may be present that are not detectable by cytological analysis
(13). Thus biological systems may have evolved very sensitive detection
systems for DNA double-stranded breaks;
-H2AX formation may function
in such a system.
There are several types of hypotheses concerning how the
-H2AX
molecules on these large amounts of chromatin might be arranged relative to the DNA double-stranded breaks. In the first type, H2AX
molecules on the strands contiguous to the breaks could be
-phosphorylated starting near the break and progressing away. The
-H2AX molecules may then provide binding sites for new components involved in recognition or repair or alternatively may lead to conformational changes in the chromatin conducive to DNA repair and
cell survival. In the crystallographic structure of the nucleosome (3),
H2A lysine 119 is situated at the edge of the nucleosome where the DNA
double helices enter and exit. An extended peptide of 20 amino acid
residues from lysine 119 to serine 139 has a maximum length of 6 nm,
while the nucleosome particle itself has a diameter of 11 nm. Thus H2AX
serine 139 has a potential range the covers part of the nucleosome and
the histone H1-containing spacer region between nucleosomes. Being on
the exterior of the nucleosome would make H2AX residue serine 139 easily accessible to kinases. If kinases tracked along the DNA about
equal distances from a DNA double-stranded break in the various cell
lines before falling off or encountering a barrier, then about equal
percentages of
-H2AX would be formed irrespective of relative H2AX
content. This model provides an explanation why a constant percentage
and not a constant number of the H2AX molecules are
-phosphorylated per Gy in various cells.
It is not necessary to postulate chromatin structures of megabase
dimensions, but there is evidence supporting chromatin structures of
this size. Yokota et al. (43), measuring the physical
distance between probes of known separation along the DNA in interphase nuclei, reported a discontinuity between the physical and genomic distances at about 2 × 106 bp, indicating that an
underlying chromatin structure of this size may be present. In
addition, Yunis (44) using preparations of midprophase human
chromosomes stained with Giemsa was able to discern about 2000 bands/haploid complement; those values yield an average size of
1.5 × 106 bp/band.
In a second type of hypothesis, the DNA double-stranded break would
still be the initiating point of the
-H2AX formation, but the
activity would diffuse away from the break in three-dimensional space.
Thus, H2AX molecules on strands of chromatin near the DNA double-stranded break would become
-phosphorylated even if those strands were on different chromosomes. In a third type of hypothesis, H2AX molecules at random throughout the nucleus would become
phosphorylated in a manner dependent on the amount of radiation.
Determining the spatial relationship between the triggering lesion and
the
-H2AX will be useful in elucidating their functional
relationship. Antibodies specific to
-H2AX will be useful in
determining the spatial characteristics of the response.
We gratefully acknowledge Dr. Kurt Kohn for
continuing support. Dr. Joan Turner kindly donated the M059J human
mutant cell line, and Dr. Gloria Li kindly donated a Ku80
/
cell
line.