ORIGINAL ARTICLE Live imaging of induced and controlled DNA double-strand break formation reveals extremely low repair by homologous recombination in human cells OD Shahar, EVS Raghu Ram, E Shimshoni, S Hareli, E Meshorer and M Goldberg The Department of Genetics, The Institute of Life Sciences, The Hebrew University, Jerusalem, Israel 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 endonu- clease-induced DSBs are rarely repaired by HR, whereas persistent DSBs result in the published HRR frequencies (non-significant HR frequency versus frequency of B10%, 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. Oncogene advance online publication, 21 November 2011; doi:10.1038/onc.2011.516 Keywords: DNA double-strand breaks; DNA repair; homologous recombination; genomic stability; ISceI Introduction DNA double-strand breaks (DSBs) are the most severe form of DNA damage since they may result in genomic instability, which is a hallmark of cancer cells. The two major pathways that repair DSBs are non-homologous end joining (NHEJ) and homologous recombination (HR). In mammalian cells, NHEJ, which is based on rejoining of juxtaposed ends, predominates throughout the cell cycle and can lead to inaccurate repair. HR repair (HRR), which relies on a homologous DNA molecule, usually the sister chromatid, as a template, is considered accurate and is restricted to S, G2 and M phases of the cell cycle (Takata et al., 1998; Rothkamm et al., 2003; Saleh-Gohari and Helleday, 2004; Mao et al., 2008; Shrivastav et al., 2008; Hartlerode and Scully, 2009). In addition to being essential for DSB repair and for the maintenance of genomic stability, HR is important for directed gene targeting in gene therapy (Yanez and Porter, 1998). HRR in human cells is widely studied using systems that are based on the combination of the overexpression of ISceI endonuclease and a reporter cassette. The commonly used DR-GFP reporter cassette contains the ISceI recognition sequence, which is absent in the human genome. A functional GFP is gained only if the ISceI-induced DSB is repaired by HR. Many discoveries were revealed using such systems, including finding HRR genes, the importance of the cell-cycle phase on the balance between HRR and NHEJ and the estimation that HRR frequency is 2–15% (Takata et al., 1998; Pierce et al., 1999; Rothkamm et al., 2003; Saleh-Gohari and Helleday, 2004; Sartori et al., 2007; Mao et al., 2008; Shrivastav et al., 2008; Hartlerode and Scully, 2009). It was suggested that repair of the ISceI-induced DSB in the DR-GFP cassette by NHEJ can destroy the ISceI site (Nakanishi et al., 2005). However, the ISceI-induced DSB can also be repaired by error-free NHEJ, which restores the ISceI site. Thus, the DSB may repeatedly occur and be repaired as long as the endonuclease remains in the nucleus (Honma et al., 2007; Mao et al., 2008; Bennardo et al., 2009). Such persistent DSBs may present a bias for data obtained using this system, which potentially allows an accurate repair of the initial DSB by NHEJ and repeated cycles of cleavage and repair. Hence, an HRR product may represent the repair of a DSB formed at later time points rather than the repair of the initial break. In addition, the described systems rely on the transfection efficiencies of an ISceI-expressing plasmid, resulting in a delay between the time of the transfection and the expression of an Received 17 May 2011; revised and accepted 11 October 2011 Correspondence: Dr E Meshorer or Dr M Goldberg, The Department of Genetics, The Institute of Life Sciences, The Hebrew University, Givat Ram, Jerusalem 91904, Israel. E-mail: meshorer@cc.huji.ac.il or goldbergm@vms.huji.ac.il Oncogene (2011) 1–10 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc