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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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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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gkt255-F4: 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.

Mentions: Next, we validated our combined dual repair assay and inducible I-SceI system in a focused study of key DSB repair genes. In this study, we sought to probe the relative roles of selected genes in both mutagenic NHEJ and HR repair, using a system that could uniquely detect dynamic shifts between the two pathways. U2OS EJ-DRs cells were nucleofected with siRNAs targeting selected DSB repair genes, followed by incubation between 48 and 72 h to allow time for downregulation of target gene expression. In parallel, cells were treated with siRNAs targeting either a scrambled sequence or a housekeeping gene (e.g. Glyceraldehyde 3-phosphate dehydrogenase; GAPDH) as negative controls. Cells were then treated with Shield1 and TA for 24 h to induce DSBs, followed by washing to remove ligands and analysis by flow cytometry after an additional 96 h. The percentages of DsRed+ and GFP+ cells transfected with siRNAs targeting the DSB repair genes were normalized to the percentages observed in cells transfected with control siRNAs for both mutagenic NHEJ and HR, respectively. In this manner, the data can be presented as a relative assessment of DSB repair on a 2D plot (which we refer to as a RADAR plot), which allows one to rapidly assess the effects of a given siRNA knockdown on both mutagenic NHEJ and HR repair activity. A schematic of how these data are presented is shown in Figure 4A.Figure 4.


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)

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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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675474&req=5

gkt255-F4: 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.
Mentions: Next, we validated our combined dual repair assay and inducible I-SceI system in a focused study of key DSB repair genes. In this study, we sought to probe the relative roles of selected genes in both mutagenic NHEJ and HR repair, using a system that could uniquely detect dynamic shifts between the two pathways. U2OS EJ-DRs cells were nucleofected with siRNAs targeting selected DSB repair genes, followed by incubation between 48 and 72 h to allow time for downregulation of target gene expression. In parallel, cells were treated with siRNAs targeting either a scrambled sequence or a housekeeping gene (e.g. Glyceraldehyde 3-phosphate dehydrogenase; GAPDH) as negative controls. Cells were then treated with Shield1 and TA for 24 h to induce DSBs, followed by washing to remove ligands and analysis by flow cytometry after an additional 96 h. The percentages of DsRed+ and GFP+ cells transfected with siRNAs targeting the DSB repair genes were normalized to the percentages observed in cells transfected with control siRNAs for both mutagenic NHEJ and HR, respectively. In this manner, the data can be presented as a relative assessment of DSB repair on a 2D plot (which we refer to as a RADAR plot), which allows one to rapidly assess the effects of a given siRNA knockdown on both mutagenic NHEJ and HR repair activity. A schematic of how these data are presented is shown in Figure 4A.Figure 4.

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