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

Abstract

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 32 PO 4 incorporation on acetic acid-urea-Triton X-100-acetic acid-ureacetyltrimethylammonium bromide or SDS-acetic acidurea-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 ؋ 10 9 base pairs of a mammalian G 1 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 doublestranded break. This value, which corresponds to about 2 ؋ 10 6 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.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 bp 1 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 heterochromati...

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