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

Takahisa Furuta, Haruyuki Takemura, Zhi Yong Liao, Gregory J. Aune, Christophe Redon, Olga A. Sedelnikova, Duane R. Pilch, Emmy P. Rogakou, Arkady Celeste, Hua Tang Chen, Andre Nussenzweig, Mirit I. Aladjem, William M. Bonner, Yves Pommier

Research output: Contribution to journalArticlepeer-review

354 Scopus citations

Abstract

DNA double-strand breaks originating from diverse causes in eukaryotic cells are accompanied by the formation of phosphorylated H2AX (γH2AX) foci. Here we show that γH2AX 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 γH2AX formation can be prevented with the phosphatidylinositol 3-OH kinase-related kinase inhibitor wortmannin; however, in contrast to ionizing radiation, only camptothecin-induced γH2AX formation can be prevented with the DNA replication inhibitor aphidicolin and enhanced with the checkpoint abrogator 7-hydroxystaurosporine. This γH2AX 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 γH2AX formation at the sites of replication-mediated DNA double-strand breaks. Mre11- and Nbs1-deficient cells are still able to form γH2AX. 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 γH2AX response for double-strand breaks induced by replication fork collision. γH2AX foci are required for recruiting repair and checkpoint protein complexes to the replication break sites.

Original languageEnglish (US)
Pages (from-to)20303-20312
Number of pages10
JournalJournal of Biological Chemistry
Volume278
Issue number22
DOIs
StatePublished - May 30 2003

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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