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.
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.
Furuta
Takahisa
T
Laboratory of Molecular Pharmacology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892-4255, USA.
Takemura
Haruyuki
H
Liao
Zhi-Yong
ZY
Aune
Gregory J
GJ
Redon
Christophe
C
Sedelnikova
Olga A
OA
Pilch
Duane R
DR
Rogakou
Emmy P
EP
Celeste
Arkady
A
Chen
Hua Tang
HT
Nussenzweig
Andre
A
Aladjem
Mirit I
MI
Bonner
William M
WM
Pommier
Yves
Y
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
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