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

• Published in: Oncogene Vol. 31; no. 30; pp. 3495 - 3504
• Main Authors: Shahar, O D, Ram, E V S Raghu, Shimshoni, E, Hareli, S, Meshorer, E, Goldberg, M
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 26.07.2012; Nature Publishing Group
• Subjects: 631/337/1427/2122; 631/337/1427/2190; 631/80/2373; Apoptosis; Cancer; Cancer cells; Cell Biology; Cell cycle; Cell Cycle - physiology; Cell Line; Cell Tracking - methods; Data processing; Deoxyribonuclease I - physiology; Deoxyribonucleic acid; DNA; DNA Breaks, Double-Stranded; DNA damage; DNA repair; Double-strand break repair; Endonuclease; Genetic recombination; Genome, Human - physiology; Genomes; Genomic instability; Genomics; Homologous recombination; Homologous recombination repair; Human Genetics; Human genome; Humans; Image Processing, Computer-Assisted - instrumentation; Image Processing, Computer-Assisted - methods; imaging; Internal Medicine; Mammalian cells; Medicine; Medicine & Public Health; Microscopy - instrumentation; Microscopy - methods; Non-homologous end joining; Oncology; original-article; Physiological aspects; Recombinational DNA Repair - physiology; Time-Lapse Imaging - instrumentation; Time-Lapse Imaging - methods; Yeast; I; homologous recombination; DNA double-strand breaks; genomic stability; DNA repair
• ISSN: 0950-9232; 1476-5594
• DOI: 10.1038/onc.2011.516

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.