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p53 protects from replication-associated DNA double-strand breaks in mammalian cells

Oncogene volume 23, pages 2324–2329 (2004)Cite this article

Abstract

Genetic instability caused by mutations in the p53 gene is generally thought to be due to a loss of the DNA damage response that controls checkpoint functions and apoptosis. Cells with mutant p53 exhibit high levels of homologous recombination (HR). This could be an indirect consequence of the loss of DNA damage response or p53 could have a direct role in HR. Here, we report that p53−/− mouse embryonic fibroblasts (MEFs) exhibit higher levels of the RAD51 protein and increased level of spontaneous RAD51 foci Agents that stall replication forks, for example, hydroxyurea (HU), potently induce HR repair and RAD51 foci. To test if the increase in RAD51 foci in p53−/− MEFs was due to an increased level of damage during replication, we measured the formation of DNA double-strand breaks (DSBs) in p53+/+ and p53−/− MEFs following treatments with HU. We found that HU induced DSBs only in p53−/− MEFs, indicating that p53 is involved in a pathway to protect stalled replication forks from being collapsed into a substrate for HR. Also, p53 is upregulated in response to agents that inhibit DNA replication, which supports our hypothesis. Finally, we observed that the DSBs produced in p53−/− MEFs did not result in a permanent arrest of replication and that they were repaired. Altogether, we suggest that the effect of p53 on HR and RAD51 levels and foci can be explained by the idea that p53 suppresses formation of recombinogenic lesions.

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Abbreviations

CPT:

camptothecin

DSBs:

DNA double-strand breaks

HR:

homologous recombination

HU:

hydroxyurea

MEFs:

mouse embryonic fibroblasts

TdR:

thymidine

VP16:

etoposide

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Acknowledgements

We thank Miss Elena Lopez for technical assistance, Drs Mark Meuth, Helen Bryant and Kayan Parker for critically reading this manuscript and Dr Agneta Önfelt for the use of microscopy. This investigation was supported by the Swedish Cancer Society, the Swedish National Board for Laboratory Animals, Biological & Biotechnological Sciences Research Council and Yorkshire Cancer Research.

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Authors and Affiliations

  1. The Institute for Cancer Studies, University of Sheffield, Medical School, Beech Hill Road, Sheffield, S10 2RX, UK

    Anuradha Kumari & Thomas Helleday

  2. Department of Genetic and Cellular Toxicology, Arrhenius Laboratory, Stockholm University, Stockholm, S-106 91, Sweden

    Niklas Schultz & Thomas Helleday

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  1. Anuradha Kumari
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Correspondence to Thomas Helleday.

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Kumari, A., Schultz, N. & Helleday, T. p53 protects from replication-associated DNA double-strand breaks in mammalian cells. Oncogene 23, 2324–2329 (2004). https://doi.org/10.1038/sj.onc.1207379

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