Analysis of Nonhomologous End Joining and Homologous Recombination Efficiency in HEK-293T Cells Using GFP-Based Reporter Systems
Summary
This protocol describes an extrachromosomal nonhomologous end joining (NHEJ) assay and homologous recombination (HR) assay to quantify the efficiency of NHEJ and HR in HEK-293T cells.
Abstract
DNA double-strand breaks (DSBs) represent the most perilous DNA lesions, capable of inducing substantial genetic information loss and cellular demise. In response, cells employ two primary mechanisms for DSB repair: nonhomologous end joining (NHEJ) and homologous recombination (HR). Quantifying the efficiency of NHEJ and HR separately is crucial for exploring the relevant mechanisms and factors associated with each. The NHEJ assay and HR assay are established methods used to measure the efficiency of their respective repair pathways. These methods rely on meticulously designed plasmids containing a disrupted green fluorescent protein (GFP) gene with recognition sites for endonuclease I-SceI, which induces DSBs. Here, we describe the extrachromosomal NHEJ assay and HR assay, which involve co-transfecting HEK-293T cells with EJ5-GFP/DR-GFP plasmids, an I-SceI expressing plasmid, and an mCherry expressing plasmid. Quantitative results of NHEJ and HR efficiency are obtained by calculating the ratio of GFP-positive cells to mCherry-positive cells, as counted by flow cytometry. In contrast to chromosomally integrated assays, these extrachromosomal assays are more suitable for conducting comparative investigations involving multiple established stable cell lines.
Introduction
A DNA double-strand break (DSB) is the most deleterious form of DNA damage, potentially leading to genome instability, chromosomal rearrangements, cellular senescence, and cell death if not repaired promptly1. Two well-established pathways, nonhomologous end joining (NHEJ) and homologous recombination (HR), are recognized for their effectiveness in addressing DNA DSBs2,3. HR is considered an error-free mechanism for DSB repair, utilizing homologous sequences in the sister chromatid as a template to restore the original configuration of the injured DNA molecule3. NHEJ, on the other hand, is an error-prone DSB repair pathway that joins the broken DNA ends without relying on any template2.
The NHEJ assay and HR assay are classical methods originally developed in Jasin's laboratory at Memorial Sloan-Kettering Cancer Center and utilized to quantify the efficiency of NHEJ and HR, respectively4,5,6,7. These assays play a crucial role in investigating the relevant mechanisms and factors associated with NHEJ and HR8,9,10,11,12,13,14. Both assays rely on the implementation of two disrupted GFP reporters, EJ5-GFP and DR-GFP, to monitor the repair of DSBs induced by the I-SceI endonuclease. The EJ5-GFP reporter is employed in the NHEJ assay, while the DR-GFP reporter is utilized in the HR assay. Each reporter is subtly designed so that the I-SceI-induced DSBs can only be repaired by a specific repair pathway to restore a GFP expression cassette4,5.
The NHEJ assay and HR assay can be conducted using either a chromosomally integrated or an extrachromosomal approach15,16. The chromosomally integrated approach necessitates the integration of the disrupted GFP reporters into the genome, allowing the analysis of DSB repair within a chromosomal context6,15. However, this approach requires prolonged cell passaging and is unsuitable for comparative studies involving multiple cell lines due to arbitrary chromosomal integration, introducing an additional confounding factor apart from inherent differences. In this protocol, we describe the extrachromosomal NHEJ assay and HR assays, involving the transient transfection of the disrupted GFP and I-SceI plasmids into HEK-293T cells, followed by flow cytometry analysis (the experiment workflow is shown in Figure 1). These non-integrated reporter assays were originally reported by Jasin's laboratory to study DNA interstrand cross-links repair16 and have been employed to assess NHEJ efficiency and HR efficiency by several laboratories9,10,11,12,13,14,17,18,19, including ours11. These extrachromosomal approaches facilitate the analysis of DSB repair in comparative studies involving multiple established stable cell lines.
Protocol
Representative Results
Discussion
The method described here has been employed in several papers to assess NHEJ efficiency and HR efficiency9,10,11,12,13,14,16,17,18,19. This method is pertinent for elucidating the underly…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
This research was funded by the Natural Science Foundation of Heilongjiang Province of China (YQ2022C036) and the Graduate Innovation Foundation of Qiqihar Medical University (QYYCX2022-06). Figure 1 produced using MedPeer.
Materials
| 6 cm dishes | BBI | F611202-9001 | |
| 6 well plates | Corning | 3516 | |
| Ampicillin | Beyotime | ST007 | Working concentration: 100 μg/mL |
| DH5α Competent Cells | TIANGEN | CB101 | |
| DMEM | Hyclone | SH30022.01 | |
| DR-GFP | Addgene | 26475 | |
| EJ5-GFP | Addgene | 44026 | |
| EndoFree Maxi Plasmid kit | TIANGEN | DP117 | alternative endotoxin-free plasmid extraction kit can be used |
| FACS tubes | FALCON | 352054 | |
| Fetal bovine serum | CLARK | FB25015 | |
| Flow cytometer | BD Biosciences | BD FACSCalibur | |
| FlowJo V.10.1 | Treestar | alternative analysis software can be used | |
| HEK-293T cells | National Infrastructure of Cell Line Resource | 1101HUM-PUMC000091 | |
| Lipo3000 | Invitrogen | L3000015 | alternative transfection regents can be used |
| PBS | Biosharp | BL601A | |
| pCBASceI | Addgene | 26477 | I-SceI expressing plasmid |
| PCI2-HA-mCherry | alternative plasmids containing DsRed can be used | ||
| Trypsin | Gibco | 25200-056 |
Referenzen
- Huang, R., Zhou, P. K. DNA damage repair: historical perspectives, mechanistic pathways and clinical translation for targeted cancer therapy. Signal Transduct Target Ther. 6 (1), 254 (2021).
- Pannunzio, N. R., Watanabe, G., Lieber, M. R. Nonhomologous DNA end-joining for repair of DNA double-strand breaks. J Biol Chem. 293 (27), 10512-10523 (2018).
- Wright, W. D., Shah, S. S., Heyer, W. -. D. Homologous recombination and the repair of DNA double-strand breaks. J Biol Chem. 293 (27), 10524-10535 (2018).
- Pierce, A. J., Johnson, R. D., Thompson, L. H., Jasin, M. XRCC3 promotes homology-directed repair of DNA damage in mammalian cells. Genes Dev. 13 (20), 2633-2638 (1999).
- Bennardo, N., Cheng, A., Huang, N., Stark, J. M. Alternative-NHEJ Is a mechanistically distinct pathway of mammalian chromosome break repair. PLoS Genet. 4 (6), e1000110 (2008).
- Gunn, A., Stark, J. M. I-SceI-based assays to examine distinct repair outcomes of mammalian chromosomal double strand breaks. Methods Mol Biol. 920, 379-391 (2012).
- Zuo, N., et al. Detection of alternative end-joining in HNSC cell lines using DNA Double-strand break reporter assays. Bio Protoc. 12 (17), 4506 (2022).
- Schrank, B. R., et al. Nuclear ARP2/3 drives DNA break clustering for homology-directed repair. Nature. 559 (7712), 61-66 (2018).
- Lu, H., Saha, J., Beckmann, P. J., Hendrickson, E. A., Davis, A. J. DNA-PKcs promotes chromatin decondensation to facilitate initiation of the DNA damage response. Nucleic Acids Res. 47 (18), 9467-9479 (2019).
- Xu, R., et al. hCINAP regulates the DNA-damage response and mediates the resistance of acute myelocytic leukemia cells to therapy. Nat Commun. 10 (1), 3812 (2019).
- Wang, T., et al. WASH interacts with Ku to regulate DNA double-stranded break repair. iScience. 25 (1), 103676 (2022).
- Guo, G., et al. Reciprocal regulation of RIG-I and XRCC4 connects DNA repair with RIG-I immune signaling. Nat Commun. 12 (1), 2187 (2021).
- Watkins, J., et al. Genomic complexity profiling reveals that HORMAD1 overexpression contributes to homologous recombination deficiency in triple-negative breast cancers. Cancer Discov. 5 (5), 488-505 (2015).
- Chen, Y., et al. USP44 regulates irradiation-induced DNA double-strand break repair and suppresses tumorigenesis in nasopharyngeal carcinoma. Nat Commun. 13 (1), 501 (2022).
- Seluanov, A., Mao, Z., Gorbunova, V. Analysis of DNA double-strand break (DSB) repair in mammalian cells. JoVE. (43), e2002 (2010).
- Nakanishi, K., Cavallo, F., Brunet, E., Jasin, M., Tsubouchi, H. . DNA recombination: Methods and Protocols. , 283-291 (2011).
- Cavallo, F., et al. Reduced proficiency in homologous recombination underlies the high sensitivity of embryonal carcinoma testicular germ cell tumors to cisplatin and poly (ADP-Ribose) polymerase inhibition. PLoS One. 7 (12), e51563 (2012).
- Unfried, J. P., et al. Long noncoding RNA NIHCOLE promotes ligation efficiency of DNA double-strand breaks in hepatocellular carcinoma. Cancer Res. 81 (19), 4910-4925 (2021).
- Cavallo, F., Caggiano, C., Jasin, M., Barchi, M., Bagrodia, A., Amatruda, J. F. . Testicular Germ Cell Tumors: Methods and Protocols. , 113-123 (2021).
- Green, M. R., Sambrook, J. The Hanahan method for preparation and transformation of competent Escherichia coli: high-efficiency transformation. Cold Spring Harbor Protocols. 2018 (3), 101188 (2018).
- Stark, J. M., Pierce, A. J., Oh, J., Pastink, A., Jasin, M. Genetic steps of mammalian homologous repair with distinct mutagenic consequences. Mol Cell Biol. 24 (21), 9305-9316 (2004).
