title: 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 gamma, these components are formed in vivo by several procedures that introduce double-stranded breaks into DNA. gamma-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. gamma-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 gamma-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 x 10(9) base pairs of a mammalian G1 genome, leads to the gamma-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 x 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, gamma-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.
authors.affiliation: Laboratory of Molecular Pharmacology, Division of Basic Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: E P
authors.initials: EP
authors.lastname: Rogakou
authors.forename: D R
authors.initials: DR
authors.lastname: Pilch
authors.forename: A H
authors.initials: AH
authors.lastname: Orr
authors.forename: V S
authors.initials: VS
authors.lastname: Ivanova
authors.forename: W M
authors.initials: WM
authors.lastname: Bonner
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D002843:Chromatin / Q000378:metabolism; D004247:DNA / Q000528:radiation effects*; D015180:Electrophoresis, Gel, Two-Dimensional; D005720:Gamma Rays; D006657:Histones / Q000378:metabolism*; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D011494:Protein Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014916:Whole-Body Irradiation
chemical_list: D002843:Chromatin; D006657:Histones; D009687:Nuclear Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010768:Phosphoserine; D004247:DNA; D011494:Protein Kinases