Assaying double-strand break repair pathway choice in mammalian cells using a targeted endonuclease or the RAG recombinase

Abstract

DNA damage repair is essential for the maintenance of genetic integrity in all organisms. Unrepaired or imprecisely repaired DNA can lead to mutagenesis, cell death, or malignant transformation. DNA damage in the form of double-strand breaks (DSBs) can occur as a result of both exogenous insults, such as ionizing radiation and drug therapies, and normal metabolic processes including V(D)J recombination. Mammalian cells have multiple pathways for repairing DSBs, including nonhomologous end-joining (NHEJ), homologous recombination (HR), and single-strand annealing (SSA). This chapter describes the use of reporter substrates for assaying the contributions of these pathways to DSB repair in mammalian cells, in particular murine embryonic stem cells. The individual contributions of NHEJ, HR, and SSA can be quantified using fluorescence and PCR-based assays after the precise introduction of DSBs either by the I-SceI endonuclease or by the RAG recombinase. These reporters can be used to assess the effects of genetic background, dominant-negative constructs, or physiological conditions on DSB repair in a wide variety of mammalian cells.

Figure 1

Fluorescence and PCR assays to measure different pathways of repair of an I-SceI endonuclease generated DSB. A. After expression of I-SceI in cells containing the DR-GFP reporter, repair of the DSB can proceed through HR, NHEJ, or SSA. Cells that repair by HR through a short-tract gene conversion without crossing-over become GFP+. The different pathways of repair can be distinguished by PCR amplification and digestion with I-SceI and/or BcgI. B. Example of the PCR site loss assay from cells containing the DR-GFP reporter transfected with empty vector or I-SceI-expression vector. The 725 bp band in the I-SceI-digest represents the product amplified from cells that have undergone HR, SSA or imprecise NHEJ. The 546 bp band in the BcgI digest represents the product amplified from cells that have undergone HR and SSA. The 725 bp band in the ISceI+ BcgI-digest represents the product amplified from cells that have undergone imprecise NHEJ. C. PCR strategy for evaluating repair by SSA. D. Fluorescence substrate SA-GFP for evaluating repair by SSA (Stark et al., 2004). Although other pathways can give rise to a GFP+ gene, they appear to be rare. (See Nakanishi et al., 2005 for using resistance to puromycin to remove long-tract gene conversion events.) Abbreviations: 1, DRGFP1; 2, DRGFP2; HR, homologous recombination, NHEJ, nonhomologous end-joining; SSA, single-strand annealing; 3A, SAGFP3A; 3B, SAGFP3B. Arrows, PCR primers.

Figure 2

Fluorescence and PCR assays to measure different pathways of repair of RAG-recombinase generated DSBs using the DRGFP-SE and DRGFP-CE reporters. A. RAG-induced excision of the sequence between the RSS elements (light gray) in DRGFP-SE and DRGFP-CE results in DSB ends that can undergo repair by NHEJ (i.e., V(D)J recombination), HR, or SSA. For DRGFP-SE, cleavage produces two blunt, chromosomal signal ends. For DRGFP-CE, cleavage produces two hairpin, chromosomal coding ends. As for DR-GFP, HR using the downstream repair template results in a GFP+ cell. B. A representative PCR-Southern using different mixtures of genomic DNA from untransfected cells containing the DRGFP (1.1 kb band) and DRGFP-CE (1.5 kb band) reporters. The lower band is overrepresented due to unequal PCR amplification of the two products. C. A representative PCR-Southern demonstrating a combination of HR and V(D)J recombination of approximately 1–4% in wildtype cells. Because the percent of GFP+ cells is typically 0.02–0.1%, the vast majority of this product arises from V(D)J recombination in wildtype cells. D. PCRSouthern assay for SSA demonstrating approximately 100-fold higher amplification for cells containing the DR-GFP reporter after I-SceI expression compared to cells containing the DRGFP-SE or DRGFP-CE reporters after RAG expression. Arrows, PCR primers.