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 language | English (US) |
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Pages (from-to) | 20303-20312 |
Number of pages | 10 |
Journal | Journal of Biological Chemistry |
Volume | 278 |
Issue number | 22 |
DOIs | |
State | Published - May 30 2003 |
Externally published | Yes |
ASJC Scopus subject areas
- Molecular Biology
- Biochemistry
- Cell Biology