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Efficient rejoining of radiation-induced DNA double-strand breaks in vertebrate cells deficient in genes of the RAD52 epistasis group

Oncogene volume 20, pages 2212–2224 (2001)Cite this article

Abstract

Rejoining of ionizing radiation (IR) induced DNA DSBs usually follows biphasic kinetics with a fast (t50: 5–30 min) component attributed to DNA-PK-dependent non-homologous endjoining (NHEJ) and a slow (t50: 1–20 h), as of yet uncharacterized, component. To examine whether homologous recombination (HR) contributes to DNA DSB rejoining, a systematic genetic study was undertaken using the hyper-recombinogenic DT40 chicken cell line and a series of mutants defective in HR. We show that DT40 cells rejoin IR-induced DNA DSBs with half times of 13 min and 4.5 h and contributions by the fast (78%) and the slow (22%) components similar to those of other vertebrate cells with 1000-fold lower levels of HR. We also show that deletion of RAD51B, RAD52 and RAD54 leaves unchanged the rejoining half times and the contribution of the slow component, as does also a conditional knock out mutant of RAD51. A significant reduction (to 37%) in the contribution of the fast component is observed in Ku70−/− DT40 cells, but the slow component, operating with a half time of 18.4 h, is still able to rejoin the majority (63%) of DSBs. A double mutant Ku70−/−/RAD54−/− shows similar half times to Ku70−/− cells. Thus, variations in HR by several orders of magnitude leave unchanged the kinetics of rejoining of DNA DSBs, and fail to modify the contribution of the slow component in a way compatible with a dependence on HR. We propose that, in contrast to yeast, cells of vertebrates are ‘hard-wired’ in the utilization of NHEJ as the main pathway for rejoining of IR-induced DNA DSBs and speculate that the contribution of homologous recombination repair (HRR) is at a stage after the initial rejoining.

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Acknowledgements

Supported by NCI Grants CA42026 and P30-CA56036 awarded from NIH, DHHS. Special thanks go to Ms Nancy Mott for help in the preparation of the manuscript.

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Authors and Affiliations

  1. Department of Radiation Oncology of Kimmel Cancer Center, Jefferson Medical College, Thompson Building Room B-1, Philadelphia, 19107, Pennsylvania, PA, USA

    Huichen Wang, Zhao-Chong Zeng, Tu-Anh Bui & George Iliakis

  2. Bayer-chair Department of Molecular Immunology and Allergology, Faculty of Medicine, Kyoto University, Konoe Yoshida, 606-8501, Sakyo-ku, Kyoto, Japan

    Minoru Takata & Shunichi Takeda

  3. CREST, JST (Japan Science and Technology),

    Eiichiro Sonoda

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Wang, H., Zeng, ZC., Bui, TA. et al. Efficient rejoining of radiation-induced DNA double-strand breaks in vertebrate cells deficient in genes of the RAD52 epistasis group. Oncogene 20, 2212–2224 (2001). https://doi.org/10.1038/sj.onc.1204350

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