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Live imaging of induced and controlled DNA double-strand break formation reveals extremely low repair by homologous recombination in human cells

Oncogene volume 31, pages 3495–3504 (2012)Cite this article

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Abstract

DNA double-strand breaks (DSBs), the most hazardous DNA lesions, may result in genomic instability, a hallmark of cancer cells. The main DSB repair pathways are non-homologous end joining (NHEJ) and homologous recombination (HR). In mammalian cells, NHEJ, which can lead to inaccurate repair, predominates. HR repair (HRR) is considered accurate and is restricted to S, G2 and M phases of the cell cycle. Despite its importance, many aspects regarding HRR remain unknown. Here, we developed a novel inducible on/off switch cell system that enables, for the first time, to induce a DSB in a rapid and reversible manner in human cells. By limiting the duration of DSB induction, we found that non-persistent endonuclease-induced DSBs are rarely repaired by HR, whereas persistent DSBs result in the published HRR frequencies (non-significant HR frequency versus frequency of ∼10%, respectively). We demonstrate that these DSBs are repaired by an accurate repair mechanism, which is distinguished from HRR (most likely, error-free NHEJ). Notably, our data reveal that HRR frequencies of endonuclease-induced DSBs in human cells are >10-fold lower than what was previously estimated by prevailing methods, which resulted in recurrent DSB formation. Our findings suggest a role for HRR mainly in repairing challenging DSBs, in contrast to uncomplicated lesions that are frequently repaired by NHEJ. Preventing HR from repairing DSBs in the complex and repetitive human genome probably has an essential role in maintaining genomic stability.

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Acknowledgements

We thank members of our laboratories, Sagiv Shifman and Liran Carmel for discussions, Naomi Melamed-Book for assistance in confocal analyses, Maya Schuldiner and Yifat Cohen for technical help. Stephan P Jackson, Tom Misteli, Amir Eden and Agnes Klochendler-Yeivin for reagents, Ilana Livyatan for critical reading of the manuscript, Evi Soutoglou for discussions and reagents, and the Edmond J Safra Foundation for financial support. MG is supported by the AFHU, The United States Binational Science Foundation (2009286) and the Israel Cancer Association. EM is a Joseph H and Belle R Braun senior lecturer in life sciences and is supported by the Israel Science Foundation (943/09), the Israel Cancer Research Foundation, the Israel Ministry of Health (6007), the European Union (IRG-206872 and 238176), the Internal Applicable Medical Grants of the HU and the Israel Psychobiology Institute. EVSRR is supported by the Lady Davies Fellowship program.

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Authors and Affiliations

  1. The Department of Genetics, The Institute of Life Sciences, The Hebrew University, Jerusalem, Israel

    O D Shahar, E V S Raghu Ram, E Shimshoni, S Hareli, E Meshorer & M Goldberg

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  1. O D Shahar
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  2. E V S Raghu Ram
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  3. E Shimshoni
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  4. S Hareli
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  5. E Meshorer
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  6. M Goldberg
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Correspondence to E Meshorer or M Goldberg.

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Supplementary Information accompanies the paper on the Oncogene website

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Shahar, O., Ram, E., Shimshoni, E. et al. Live imaging of induced and controlled DNA double-strand break formation reveals extremely low repair by homologous recombination in human cells. Oncogene 31, 3495–3504 (2012). https://doi.org/10.1038/onc.2011.516

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