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. 2013 Jun;41(11):e115.
doi: 10.1093/nar/gkt255. Epub 2013 Apr 12.

Development of an Assay to Measure Mutagenic Non-Homologous End-Joining Repair Activity in Mammalian Cells

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Free PMC article

Development of an Assay to Measure Mutagenic Non-Homologous End-Joining Repair Activity in Mammalian Cells

Ranjit S Bindra et al. Nucleic Acids Res. .
Free PMC article

Abstract

Double-strand break (DSB) repair pathways are critical for the maintenance of genomic integrity and the prevention of tumorigenesis in mammalian cells. Here, we present the development and validation of a novel assay to measure mutagenic non-homologous end-joining (NHEJ) repair in living cells, which is inversely related to canonical NHEJ and is based on the sequence-altering repair of a single site-specific DSB at an intrachromosomal locus. We have combined this mutagenic NHEJ assay with an established homologous recombination (HR) assay such that both pathways can be monitored simultaneously. In addition, we report the development of a ligand-responsive I-SceI protein, in which the timing and kinetics of DSB induction can be precisely controlled by regulating protein stability and cellular localization in cells. Using this system, we report that mutagenic NHEJ repair is suppressed in growth-arrested and serum-deprived cells, suggesting that end-joining activity in proliferating cells is more likely to be mutagenic. Collectively, the novel DSB repair assay and inducible I-SceI will be useful tools to further elucidate the complexities of NHEJ and HR repair.

Figures

Figure 1.
Figure 1.
Initial design, development and characterization of EJ-RFP as an assay for mutagenic NHEJ repair. (A) EJ-RFP schematic depicting the cassettes from the NHEJ repair substrate and the DsRed reporter gene plasmids (Sce-TetR and TO-DsRed, respectively), which were engineered from a commercially available tet-on regulatory plasmid set (see ‘Materials and Methods’ section for details). Cleavage at the I-SceI site in the TetR plasmid can disrupt the ORF resulting in loss of TetR binding to its recognition sequence in the reporter gene plasmid, with consequent DsRed gene expression. (B) Novel FACS-based strategy to rapidly isolate cells with intrachromosomally integrated repair substrate and reporter gene plasmids. DsRed fluorescence intensity histograms are presented in a staggered offset format and in chronological order. The Sce-TetR and TO-DsRed plasmids were co-transfected into RKO cells and analyzed by flow cytometry after 2 days in culture (purple histogram), which revealed low levels of DsRed fluorescence. Cells were analyzed again after 1 week in antibiotic selection to enrich for RKO cells with intrachromosomally integrated plasmids, which revealed two distinct DsRed− and DsRed+ cell populations (brown histogram). The DsRed− population (indicated by an asterisk) was isolated by FACS and confirmed to contain predominantly DsRed− cells by flow cytometry one day after sorting (green histogram). Doxycycline was added to these cells for 24 h, which induced a DsRed+ population (red histogram; DsRed+ population indicated by double asterisks), and these cells were isolated by FACS. The isolated cells were then washed and incubated in culture medium without doxycycline for ∼1 week to allow reconstitution of DsRed repression by Sce-TetR. These cells were analyzed every 2 days to measure residual DsRed fluorescence, which steadily declined during this time (green, pink and blue histograms). Doxycycline was once again added for 24 h to these cells, which induced DsRed expression in the majority of the cells (orange histogram), indicating successful enrichment of cells containing stably integrated functional copies of the Sce-TetR and TO-DsRed plasmids. This polyclonal cell line was referred to as RKO-EJ. (C) Confirmation of doxycycline-inducible DsRed expression in RKO-EJ cells isolated in (B), as detected by flow cytometry. Graphs represent bivariate histograms of DsRed fluorescence (y-axis) versus the FL-1 channel (x-axis) to facilitate the visualization of individual cells. Inset panels show representative epifluorescence microscopy images of DsRed fluorescence in RKO EJ cells 24 h after doxycycline treatment (a representative inset panel in the −doxycycline panel shows a light microscopy image confirming high cell density, which is apparent in the +doxycycline panel). (D) Detection of DsRed+ cells after I-SceI transfection in RKO-EJ cells. RKO-EJ cells were transfected with an I-SceI expression vector to induce a DSB at the Sce-TetR locus, followed by flow cytometry analysis after 96 h. (E) Sample flow cytometry plots for DsRed− and DsRed+ cells isolated 2 weeks after I-SceI plasmid transfection. Genomic DNA was isolated from these cells for analysis of the breakpoint by PCR amplification and DNA sequencing later in the text. (F) PCR amplification and in vitro restriction digestion analysis of PCR amplicons from the breakpoint junction at the Sce-TetR locus. Schematic depicting the approximate locations of the PCR primers in the locus is shown earlier in text. Agarose gel shows complete digestion of PCR amplicon generated from Sce-TetR plasmid template DNA using I-SceI (pSce Tet lane). Similar results were obtained with genomic DNA isolated from RKO EJ cells (no DSB lane), and these two lanes serve as controls to confirm that our enzymatic conditions are optimized for complete digestion of product. The majority of the PCR amplicons generated from RFP− cell genomic DNA are cleaved by I-SceI, suggesting that minimal mutagenic NHEJ events have occurred in this cell population (RFP− lane). In contrast, the PCR amplicons from the DsRed+ cells are almost completely resistant to digestion with I-SceI, suggesting that mutagenic repair events have occurred in the DsRed+ cells. Colors on the gel have been inverted for clarity. Asterisk indicates that the two cleavage bands are closely spaced and thus may appear as a single band on the gel. (G) DNA sequence analysis and alignment of the breakpoint in individually cloned Sce-TetR alleles isolated from unsorted cells after I-SceI plasmid transfection to induced DSBs. The consensus I-SceI sequence and site of cleavage in the Sce-TetR locus is shown for reference (highlighted in red and blue text). Sequence deletions are indicated by a dashed line, sequence insertions are indicated in green text and regions of microhomology are underlined for reference.
Figure 2.
Figure 2.
Creation of other EJ-RFP cell lines and combination with DR-GFP, an assay for homologous recombination. (A) Introduction of the EJ-RFP system into the glioma cell line, M059K, using the FACS-based enrichment approach described in Figure 1B. Representative flow cytometry plots are shown: low levels of DsRed+ cells in log-phase M059K-EJ cells (left), with high levels of DsRed+ cells after doxycycline exposure (middle) and a substantial increase in the percentages of DsRed+ cells are observed after transfection with an I-SceI plasmid (right). (B) Schematic of the previously described DR-GFP assay to measure HR in cells at an intrachromosomal site (21). (C) Schematic of the readout that would be obtained from a cell line containing integrated copies of both the EJ-RFP and DR-GFP assays, in which mutagenic NHEJ (mNHEJ) is detected as an increase in DsRed+ cells and HR is detected as in increase in GFP+ cells. The assays each report on DSB repair activities at separate loci; thus, it would be possible to detect cells in which both mNHEJ and HR repair events have occurred (which would appear as DsRed+ and GFP+, respectively). (D) Creation of U2OS EJ-DR cells, containing stable copies of both DSB repair assays as described in (C). A bivariate flow cytometry histogram of DsRed fluorescence (y-axis) versus GFP fluorescence (x-axis) is shown representing mNHEJ and HR, respectively, 96 h after transfection with an I-SceI plasmid to induce DSBs. This cell line was created using the FACS-based enrichment approach described in Figure 1B. A substantial increase in the percentages of both DsRed+ and GFP+ cells is observed after I-SceI plasmid transfection, indicating robust levels of mutagenic NHEJ and HR repair, respectively. Low levels of DsRed/GFP+ cells were observed in the absence of DSB induction, as shown later in (E). (E) Isolation of a U2OS EJ-DR single cell clone with low background levels of fluorescent protein expression (left), which is inducible on I-SceI plasmid transfection followed by analysis after 96 h (right). (F) Time course analysis of the levels of DsRed+ and GFP+ cells after I-SceI plasmid transfection in a representative U2OS EJ-DR single cell clone, corresponding to mNHEJ and HR repair, respectively, as detected by flow cytometry.
Figure 3.
Figure 3.
Development of a novel system for ligand-inducible cleavage by I-SceI. (A) Creation of a novel I-SceI fusion protein with an N-terminal ligand-dependent dd and a C-terminal GR ligand-binding domain, which we have named ddSceGR (schematic shown in top). These domains induce protein stability and nuclear localization in the presence of the ligands Shield1 and TA, respectively. Induction of ddSceGR protein expression levels was confirmed by western blot analysis with an antibody specific to the dd (lower). SMC1 is shown as a loading control for reference. (B) Confirmation of ligand-induced protein stability and nuclear localization by confocal microscopy in U2OS EJ-DR cells transiently transfected with a ddSceGR plasmid in the presence or absence of Shield1 and TA ligands (1 µm and 100 nm, respectively). The ddSceGR protein was detected using an antibody specific to the GR domain, and images were taken 24 h after transfection and ligand addition. (C) Ligand-dependent control of DSB induction, as detected by HR repair in U2OS DR cells after transfection with ddSceGR. Immediately after transfection, U2OS DR cells were equally split into two plates; Shield1 and TA ligands were added to one plate (with vehicles alone added to the other plate), and the GFP+ cells were assessed by flow cytometry at 72 h. (D) Creation of a U2OS EJ-DR single cell clone containing stably integrated ddSceGR, which we have named U2OS EJ-DRs. Representative flow cytometry plots for a U2OS EJ-HRs single cell clone demonstrating low levels of DsRed+ and GFP+ cells in the absence of Shield1 and TA ligands, which is highly inducible after a 24-h exposure to the ligands. (E) Robust ligand-dependent DSB induction and consequent NHEJ repair requires both Shield1 and TA ligands. U2OS EJ-DRs cells were exposed to vehicle alone (DMSO), Shield1 alone, TA alone or both ligands for 24 h, followed by flow cytometric analysis of DsRed+ cells after 96 h to measure induced mNHEJ repair. Experiments were performed in duplicate, and error bars represent standard deviations. (F) Stability of induced DsRed+ and GFP+ cell percentages in U2OS EJ-DRs cells after a 24-h incubation with Shield1 and TA ligands, as detected by flow cytometry at 24-h intervals (starting at 96 h after ligand exposure). (G) Analysis of the effects of Shield1 and TA ligand incubation time on induced mNHEJ and HR repair rates. U2OS EJ-DRs cells were incubated with ligands for the indicated periods followed by media washing and analysis of DsRed+ and GFP+ cells after 96 h to measure induced mNHEJ and HR repair, respectively. Data points at the y-intercept reflect treatment with vehicle alone (DMSO). Experiments were performed in triplicate, and error bars represent SEM.
Figure 4.
Figure 4.
Validation of the EJ-DRs system in a focused siRNA-based study of key DSB repair genes and a growth-arrest study. (A) Schematic of data presentation format for siRNA and growth studies, referred to as a relative assessment of DSB repair (RADAR) plot. Rates of mNHEJ and HR repair, as detected by flow cytometric analysis of DsRed+ and GFP+ cells, respectively, for a given siRNA treatment or growth condition are normalized to that obtained from control cells after transfection with a scrambled siRNA sequence (indicated by black dot in center of plot). Potential effects on DSB repair pathways are shown in each quadrant for reference. (B) RADAR plot analysis of U2OS EJ-DRs cells after treatment with the indicated siRNAs or cell growth conditions. Experiments were performed in triplicate or quadruplicate, and error bars represent SEM. (C) Representative western blots from U2OS EJ-DRs cells treated with the siRNAs targeting the indicated proteins, demonstrating substantial knockdown of target genes. Cell extracts were isolated 72 h after siRNA nucleofection, and SMC1 is shown as a loading control for reference. (D) IR-induced DSB repair protein foci analysis in growth-arrested serum-deprived U2OS EJ-DRs cells. Cells were treated or not with IR (10 Gy) followed by incubation for 4 h, fixed and stained with antibodies targeting the indicated proteins and then analyzed by confocal microscopy. (E) Quantitation of DSB repair protein foci in U2OS EJ-DRs cells from (D); cells with >5 foci were scored as positive. Log-phase irradiated U2OS EJ-DR cells were also analyzed in parallel as a positive control to confirm robust foci-staining protocols. Experiments were performed in duplicate, and error bars represent standard deviations.

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