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Development of an assay to measure mutagenic non-homologous end-joining repair activity in mammalian cells.

Bindra RS, Goglia AG, Jasin M, Powell SN - Nucleic Acids Res. (2013)

Bottom Line: 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.Collectively, the novel DSB repair assay and inducible I-SceI will be useful tools to further elucidate the complexities of NHEJ and HR repair.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.

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.

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Related in: MedlinePlus

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.
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gkt255-F3: 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.

Mentions: Based on the findings discussed earlier in the text, we hypothesized that the addition of a ligand-dependent protein stability domain would provide an additional level of control to regulate DSB induction by I-SceI. A ‘two-tiered’ control of I-SceI cleavage (i.e. protein levels and nucleocytoplasmic localization) likely would facilitate the creation of cell lines with intrachromosomally integrated copies of this modified endonuclease, and we thought it may reduce the need for continuous culture in charcoal-stripped FBS. To this end, we added a destabilizing domain (dd) derived from the FKBP12 protein to the N-terminus of Sce-GR. This modified fusion protein is referred to as ddSceGR. The addition of the drug, Shield1, blocks the destabilizing effect of the N-terminal domain, resulting in a rapid increase in protein levels. This system was developed by the Wandless laboratory (47), and it is now commercially available as the ProteoTuner system (Clontech Laboratories, Inc.). As an initial test, we transiently transfected the ddSceGR vector into U2OS EJ-DR cells, followed by western blot analysis and confocal immunofluorescence microscopy in the presence or absence of the Shield1 and TA ligands. Antibodies specific to the FKBP12 and GR domains were used to detect ddSceGR expression in these studies. As shown in Figure 3A and B, ddSceGR protein levels were substantially higher in the presence of ligands, as detected by both western blot and confocal microscopy analysis. Furthermore, ddSceGR protein was predominantly localized to the nucleus in these cells after ligand addition (Figure 3B and Supplementary Figure S1F). Next, we assessed the rates of ligand-induced cleavage indirectly by measuring HR after transient transfection with ddSceGR in U2OS DR cells. Cells were incubated in ligands for 24 h immediately after ddSceGR nucleofection, followed by two washes with culture medium that did not contain TA/Shield1, and then analyzed by flow cytometry after an additional 72 h. As shown in Figure 3C, minimal percentages of GFP+ cells were detected 72 h after ddSceGR transfection in the absence of ligands (0.3% GFP+ cells). In contrast, the addition of ligands induced a significant increase in ddSceGR-induced HR activity (7.1% GFP+ cells, representing an almost 25-fold increase in repair compared with the control cells). The addition of the N-terminal dd domain reduced the overall cleavage activity of I-SceI by ∼30% compared with SceGR (Supplementary Figure S1B). However, it reduced background cleavage activity in the absence of ligands by >3-fold. Thus, the addition of both domains significantly enhanced the relative inducibility of cleavage rates under these conditions.Figure 3.


Development of an assay to measure mutagenic non-homologous end-joining repair activity in mammalian cells.

Bindra RS, Goglia AG, Jasin M, Powell SN - Nucleic Acids Res. (2013)

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.
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Related In: Results  -  Collection

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gkt255-F3: 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.
Mentions: Based on the findings discussed earlier in the text, we hypothesized that the addition of a ligand-dependent protein stability domain would provide an additional level of control to regulate DSB induction by I-SceI. A ‘two-tiered’ control of I-SceI cleavage (i.e. protein levels and nucleocytoplasmic localization) likely would facilitate the creation of cell lines with intrachromosomally integrated copies of this modified endonuclease, and we thought it may reduce the need for continuous culture in charcoal-stripped FBS. To this end, we added a destabilizing domain (dd) derived from the FKBP12 protein to the N-terminus of Sce-GR. This modified fusion protein is referred to as ddSceGR. The addition of the drug, Shield1, blocks the destabilizing effect of the N-terminal domain, resulting in a rapid increase in protein levels. This system was developed by the Wandless laboratory (47), and it is now commercially available as the ProteoTuner system (Clontech Laboratories, Inc.). As an initial test, we transiently transfected the ddSceGR vector into U2OS EJ-DR cells, followed by western blot analysis and confocal immunofluorescence microscopy in the presence or absence of the Shield1 and TA ligands. Antibodies specific to the FKBP12 and GR domains were used to detect ddSceGR expression in these studies. As shown in Figure 3A and B, ddSceGR protein levels were substantially higher in the presence of ligands, as detected by both western blot and confocal microscopy analysis. Furthermore, ddSceGR protein was predominantly localized to the nucleus in these cells after ligand addition (Figure 3B and Supplementary Figure S1F). Next, we assessed the rates of ligand-induced cleavage indirectly by measuring HR after transient transfection with ddSceGR in U2OS DR cells. Cells were incubated in ligands for 24 h immediately after ddSceGR nucleofection, followed by two washes with culture medium that did not contain TA/Shield1, and then analyzed by flow cytometry after an additional 72 h. As shown in Figure 3C, minimal percentages of GFP+ cells were detected 72 h after ddSceGR transfection in the absence of ligands (0.3% GFP+ cells). In contrast, the addition of ligands induced a significant increase in ddSceGR-induced HR activity (7.1% GFP+ cells, representing an almost 25-fold increase in repair compared with the control cells). The addition of the N-terminal dd domain reduced the overall cleavage activity of I-SceI by ∼30% compared with SceGR (Supplementary Figure S1B). However, it reduced background cleavage activity in the absence of ligands by >3-fold. Thus, the addition of both domains significantly enhanced the relative inducibility of cleavage rates under these conditions.Figure 3.

Bottom Line: 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.Collectively, the novel DSB repair assay and inducible I-SceI will be useful tools to further elucidate the complexities of NHEJ and HR repair.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.

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.

Show MeSH
Related in: MedlinePlus