Elsevier

DNA Repair

Volume 7, Issue 10, 1 October 2008, Pages 1765-1771
DNA Repair

Brief report
Comparison of nonhomologous end joining and homologous recombination in human cells

https://doi.org/10.1016/j.dnarep.2008.06.018Get rights and content

Abstract

The two major pathways for repair of DNA double-strand breaks (DSBs) are homologous recombination (HR) and nonhomologous end joining (NHEJ). HR leads to accurate repair, while NHEJ is intrinsically mutagenic. To understand human somatic mutation it is essential to know the relationship between these pathways in human cells. Here we provide a comparison of the kinetics and relative contributions of HR and NHEJ in normal human cells. We used chromosomally integrated fluorescent reporter substrates for real-time in vivo monitoring of the NHEJ and HR. By examining multiple integrated clones we show that the efficiency of NHEJ and HR is strongly influenced by chromosomal location. Furthermore, we show that NHEJ of compatible ends (NHEJ-C) and NHEJ of incompatible ends (NHEJ-I) are fast processes, which can be completed in approximately 30 min, while HR is much slower and takes 7 h or longer to complete. In actively cycling cells NHEJ-C is twice as efficient as NHEJ-I, and NHEJ-I is three times more efficient than HR. Our results suggest that NHEJ is a faster and more efficient DSB repair pathway than HR.

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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