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Caffeine inhibits homology-directed repair of I-SceI-induced DNA double-strand breaks

Abstract

We recently reported that two Chinese hamster mutants deficient in the RAD51 paralogs XRCC2 and XRCC3 show reduced radiosensitization after treatment with caffeine, thus implicating homology-directed repair (HDR) of DNA double-strand breaks (DSBs) in the mechanism of caffeine radiosensitization. Here, we investigate directly the effect of caffeine on HDR initiated by DSBs induced by a rare cutting endonuclease (I-SceI) into one of two direct DNA repeats. The results demonstrate a strong inhibition by caffeine of HDR in wild-type cells, and a substantial reduction of this effect in HDR-deficient XRCC3 mutant cells. Inhibition of HDR and cell radiosensitization to killing shows similar dependence on caffeine concentration suggesting a cause–effect relationship between these effects. UCN-01, a kinase inhibitor that effectively abrogates checkpoint activation in irradiated cells, has only a small effect on HDR, indicating that similar to radiosensitization, inhibition of checkpoint signaling is not sufficient for HDR inhibition. Recombination events occurring during treatment with caffeine are characterized by rearrangements reminiscent to those previously reported for the XRCC3 mutant, and immunofluorescence microscopy demonstrates significantly reduced formation of IR-specific RAD51 foci after caffeine treatment. In summary, our results identify inhibition of HDR as a significant contributor to caffeine radiosensitization.

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Acknowledgements

We thank Dr Maria Jasin for providing the DRaa-40 and DRirs-10 cells and the pCMV3xnls-I-SceI construt. This work was supported by NCI Grants CA42026, and CA56706 to GI and CA77693 to JAN awarded from NIH, DHHS and also supported by a grant from the Volkswagenstiftung to GI.

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Correspondence to George Iliakis.

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Wang, H., Boecker, W., Wang, H. et al. Caffeine inhibits homology-directed repair of I-SceI-induced DNA double-strand breaks. Oncogene 23, 824–834 (2004). https://doi.org/10.1038/sj.onc.1207168

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  • DOI: https://doi.org/10.1038/sj.onc.1207168

Keywords

  • caffeine
  • DNA
  • double-strand breaks (DSB)
  • repair
  • homology-directed repair
  • homologous recombination
  • nonhomologous end joining

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