title: Phosphorylation of histone H2AX and activation of Mre11, Rad50, and Nbs1 in response to replication-dependent DNA double-strand breaks induced by mammalian DNA topoisomerase I cleavage complexes.
abstract: DNA double-strand breaks originating from diverse causes in eukaryotic cells are accompanied by the formation of phosphorylated H2AX (gammaH2AX) foci. Here we show that gammaH2AX formation is also a cellular response to topoisomerase I cleavage complexes known to induce DNA double-strand breaks during replication. In HCT116 human carcinoma cells exposed to the topoisomerase I inhibitor camptothecin, the resulting gammaH2AX formation can be prevented with the phosphatidylinositol 3-OH kinase-related kinase inhibitor wortmannin; however, in contrast to ionizing radiation, only camptothecin-induced gammaH2AX formation can be prevented with the DNA replication inhibitor aphidicolin and enhanced with the checkpoint abrogator 7-hydroxystaurosporine. This gammaH2AX formation is suppressed in ATR (ataxia telangiectasia and Rad3-related) deficient cells and markedly decreased in DNA-dependent protein kinase-deficient cells but is not abrogated in ataxia telangiectasia cells, indicating that ATR and DNA-dependent protein kinase are the kinases primarily involved in gammaH2AX formation at the sites of replication-mediated DNA double-strand breaks. Mre11- and Nbs1-deficient cells are still able to form gammaH2AX. However, H2AX-/- mouse embryonic fibroblasts exposed to camptothecin fail to form Mre11, Rad50, and Nbs1 foci and are hypersensitive to camptothecin. These results demonstrate a conserved gammaH2AX response for double-strand breaks induced by replication fork collision. gammaH2AX foci are required for recruiting repair and checkpoint protein complexes to the replication break sites.
authors.affiliation: Laboratory of Molecular Pharmacology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892-4255, USA.
authors.forename: Takahisa
authors.initials: T
authors.lastname: Furuta
authors.forename: Haruyuki
authors.initials: H
authors.lastname: Takemura
authors.forename: Zhi-Yong
authors.initials: ZY
authors.lastname: Liao
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Aune
authors.forename: Christophe
authors.initials: C
authors.lastname: Redon
authors.forename: Olga A
authors.initials: OA
authors.lastname: Sedelnikova
authors.forename: Duane R
authors.initials: DR
authors.lastname: Pilch
authors.forename: Emmy P
authors.initials: EP
authors.lastname: Rogakou
authors.forename: Arkady
authors.initials: A
authors.lastname: Celeste
authors.forename: Hua Tang
authors.initials: HT
authors.lastname: Chen
authors.forename: Andre
authors.initials: A
authors.lastname: Nussenzweig
authors.forename: Mirit I
authors.initials: MI
authors.lastname: Aladjem
authors.forename: William M
authors.initials: WM
authors.lastname: Bonner
authors.forename: Yves
authors.initials: Y
authors.lastname: Pommier
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002166:Camptothecin / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000378:metabolism*; D004249:DNA Damage; D045643:DNA Repair Enzymes; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006657:Histones / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D000076228:MRE11 Homologue Protein; D051379:Mice; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D059004:Topoisomerase I Inhibitors; D014407:Tumor Cells, Cultured
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D006657:Histones; C121766:MRE11 protein, human; C512689:Mre11a protein, mouse; C112156:NBN protein, human; D009687:Nuclear Proteins; C086930:RAD50 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D059004:Topoisomerase I Inhibitors; D000076228:MRE11 Homologue Protein; D004264:DNA Topoisomerases, Type I; D045643:DNA Repair Enzymes; D002166:Camptothecin