Background

DNA double-strand breaks (DSBs) are among the most dangerous forms of DNA lesions and must be efficiently and accurately repaired to prevent genomic instability, tumorigenesis, or cell death (Rahimian et al., 2020; Huang et al., 2021). To deal with endogenous and exogenous threats, cells have evolved multiple DSB repair pathways, mainly including homologous recombination (HR), non-homologous end-joining (NHEJ), and alternative end-joining (Alt-EJ) (Mateos-Gomez et al., 2015; Liu et al., 2019; Wang et al., 2021; Fujii et al., 2022). Of these, Alt-EJ is an error-prone form of DSB repair that leads to chromosomal rearrangements and genomic instability (Liu et al., 2019; Caracciolo et al., 2021; Valikhani et al., 2021). It has been reported that Alt-EJ plays a crucial role in BRCA2-deficient cancers, human papillomavirus-associated cancers, and tumors deficient in the TGF-β signaling pathway (Ceccaldi et al., 2015; Liu et al., 2018, 2019, 2021, 2022; Guix et al., 2022). However, there are still many unknown aspects of Alt-EJ, such as the mechanisms for Alt-EJ regulation, Alt-EJ in tumor development, and DNA damage responses. To further explore the relevant mechanisms and target Alt-EJ for therapeutic benefits, the methods for Alt-EJ detection need to be established and standardized.

Reporter assays, proposed by Gunn et al. (2012), are the most commonly used tools for measuring Alt-EJ (Gunn et al., 2012). EJ2GFP-puro, a non-functional fluorescent protein expression cassette, needs to be integrated into the genome of the cells to be tested. Following the generation of a DSB in the cassette by I-SceI, the expression of the green fluorescent protein is resumed if the cell adopts Alt-EJ for repairing this DSB (Bennardo et al., 2008) (Figure 1). However, due to a lack of suitable screening conditions, the results are inevitably influenced by the efficiency of transfection. Here, we utilized another retroviral system with antibiotic resistance to make DSB and screen for target cells, a method adapted from Muraki et al. (2013). The results are presented in our recent publications (Liu et al., 2018, 2021). This method can avoid the influence of transfection efficiency on the experimental results and improve the accuracy and reproducibility of Alt-EJ study.

Our protocol has been successfully applied in the HNSC cell line SAS, as well as in the glioblastoma cell line U251 (Liu et al., 2018). Theoretically, this protocol could also be applied in other tumor or normal cell lines that require detection of Alt-EJ levels, but further validation is still needed. We can judge the mutational potential of cell chromosomes by testing the levels of Alt-EJ in sorted cells, and thus further explore the development and treatment of relevant cancers.

Figure 1. Diagram of the EJ2GFP-puro cassette. EJ2-GFP contains a GFP expression cassette that is disrupted by the I-SceI cleavage site. Repair by Alt-EJ results in restoration of GFP protein expression.