Brief reportComparison of nonhomologous end joining and homologous recombination in human cells
Introduction
Double-stranded DNA break (DSB) is a dangerous DNA lesion. If left unrepaired DSBs result in massive loss of genetic information, chromosomal aberrations, or cell death. The two major pathways for the repair of DSBs, which differ in the fidelity and template requirements, are nonhomologous end joining (NHEJ) and homologous recombination (HR) [1]. NHEJ modifies the broken DNA ends, and ligates them together with no regard for homology, generating deletions or insertions [2]. In contrast, HR uses an undamaged DNA template to repair the break, leading to the reconstitution of the original sequence [3].
Both DSB repair pathways play important roles in mammalian DSB repair [4], [5]. The exact mechanism by which NHEJ and HR interact, and how the choice is made between the two pathways remains unclear. NHEJ and HR may compete for a DSB, or the choice may be determined by the structure of the broken ends [6]. To understand human somatic mutation it is essential to know the relationship between these pathways in human cells. Surprisingly, the efficiency and kinetics of HR and NHEJ have not been directly compared.
Here we employed sensitive fluorescent reporter assays to examine NHEJ and HR in human cells. Reporter cassettes were chromosomally integrated in normal human fibroblasts immortalized by ectopic expression of telomerase, which retain all characteristics of untransformed primary cells [7]. The assays are based on the reconstitution of a functional GFP gene, where the completion of NHEJ or HR is monitored in real-time by the appearance of green cells. Using these assays we compared the kinetics of NHEJ and HR, and determined the ratio between the two processes in multiple genomic locations. We show that NHEJ is a faster and more efficient pathway than HR in human cells.
Section snippets
Cell culture
Cells were cultured at 37 °C in a 5% CO2, 3% O2 incubator, in EMEM media supplemented with 15% fetal bovine serum, 100 units/ml penicillin and 100 μg/ml streptomycin.
Construction of cell lines for detecting NHEJ and HR efficiency
To generate reporter cell lines HCA2-hTERT cells were transfected with 0.5 μg of linearized NHEJ-I, NHEJ-C, or HR reporter constructs. G418, at 1 mg/ml, was added to the media 1 day post-transfection. Colonies were picked after 8–10 days on selection. Genomic DNA was then extracted and analyzed by Southern blotting with 3′ and 5′ probes
Reporter cell lines for analysis of NHEJ and HR
To investigate the roles of NHEJ and HR in DSB repair in normal human cells we generated a series of reporter cell lines containing chromosomally integrated GFP-based reporter constructs. The reporter cassette for detecting NHEJ [8] contains a GFP gene with an artificially engineered 3 kb intron from the Pem1 gene (GFP-Pem1). The Pem1 intron contains an adenoviral exon flanked by recognition sequences for I-SceI endonuclease in direct (Fig. 1a) or inverted (Fig. 1b) orientation, which generate
Discussion
Here we report the first analysis of NHEJ and HR in normal human cells. We employ sensitive fluorescent reporter assays that allow for a direct comparison of the efficiencies of NHEJ and HR events upon induction of chromosomal DSBs with a rare-cutting endonuclease. Fluorescent assays allow for scoring DSB repair events in thousands of cells, and are highly quantitative. Furthermore, rather than relying on a single genomic locus we analyzed DSB repair at multiple chromosomal locations. By doing
Conflict of interest
None.
Acknowledgements
We thank Dara Brown, A'Shantee O'Steen, and Anna Sokolov for help with the construction of the HR reporter cell lines. This work was supported by grants from US National Institute of Health AG027237 (V.G.), American Federation for Aging Research (V.G.), the Komen Foundation (V.G.), and Ellison Medical Foundation (V.G. and A.S.).
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