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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

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.
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gkt255-F2: 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.

Mentions: A large number of assays have been developed to study both NHEJ and HR repair. Plasmid rejoining assays in transfected cells and protein extracts were used initially, and they have yielded enormous insights into DSB repair mechanisms (19). More recently, numerous assays with intrachromosomally based substrates have been developed to study NHEJ, HR and SSA repair in mammalian cells. The majority of these assays are fluorescence based and use the rare cutting endonuclease, I-SceI, to induce a single site-specific DSB in cells (20). The direct repeat green fluorescent protein (DR-GFP) assay is a commonly used assay to measure HR in living cells [schematic shown in Figure 2B (21)]. In this system, the 24-bp recognition site of I-SceI has been integrated into the GFP gene such that it disrupts the open reading frame (ORF) of the gene, and a truncated GFP gene fragment with the correct ORF sequence has been placed downstream in the construct. Repair of the cleaved I-SceI site by HR using the downstream fragment gives rise to a functional GFP gene, and GFP fluorescence then can be measured by flow cytometry. Similar GFP-based assays have been developed to measure both cNHEJ and non-canonical NHEJ in cells. Most of these systems are based on two adjacent I-SceI sites, without a downstream homology template. Simultaneous cleavage of both sites typically results in a ‘pop-out’ fragment which, depending on the orientation of the two I-SceI sites, creates either complementary or non-complementary overhangs that are exclusively repaired by NHEJ (22–25). Limitations of these current NHEJ assays include the need to induce two DSBs at a single locus, low frequencies of GFP+ cells after DSB induction, and conflicting results regarding the dependencies of various NHEJ proteins on repair activity (26,27). For example, publications have reported that Ku70/80 downregulation does not affect overall NHEJ repair frequencies using an assay based on two I-SceI sites (22), whereas other studies have suggested that loss of these proteins has a significant effect on repair rates in similarly designed NHEJ assays (24,28). Finally, DSB repair assays recently have been described in which mutagenic NHEJ repair of a nuclease-induced DSB causes loss of fluorescent protein expression via disruption of the ORF, or a specific insertion/deletion restores the ORF of a frame-shifted fluorescent protein (29–31). These findings suggest that additional assays to measure NHEJ repair are needed to further elucidate these sub-pathways and also the roles of individual DSB repair proteins.


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)

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3675474&req=5

gkt255-F2: 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.
Mentions: A large number of assays have been developed to study both NHEJ and HR repair. Plasmid rejoining assays in transfected cells and protein extracts were used initially, and they have yielded enormous insights into DSB repair mechanisms (19). More recently, numerous assays with intrachromosomally based substrates have been developed to study NHEJ, HR and SSA repair in mammalian cells. The majority of these assays are fluorescence based and use the rare cutting endonuclease, I-SceI, to induce a single site-specific DSB in cells (20). The direct repeat green fluorescent protein (DR-GFP) assay is a commonly used assay to measure HR in living cells [schematic shown in Figure 2B (21)]. In this system, the 24-bp recognition site of I-SceI has been integrated into the GFP gene such that it disrupts the open reading frame (ORF) of the gene, and a truncated GFP gene fragment with the correct ORF sequence has been placed downstream in the construct. Repair of the cleaved I-SceI site by HR using the downstream fragment gives rise to a functional GFP gene, and GFP fluorescence then can be measured by flow cytometry. Similar GFP-based assays have been developed to measure both cNHEJ and non-canonical NHEJ in cells. Most of these systems are based on two adjacent I-SceI sites, without a downstream homology template. Simultaneous cleavage of both sites typically results in a ‘pop-out’ fragment which, depending on the orientation of the two I-SceI sites, creates either complementary or non-complementary overhangs that are exclusively repaired by NHEJ (22–25). Limitations of these current NHEJ assays include the need to induce two DSBs at a single locus, low frequencies of GFP+ cells after DSB induction, and conflicting results regarding the dependencies of various NHEJ proteins on repair activity (26,27). For example, publications have reported that Ku70/80 downregulation does not affect overall NHEJ repair frequencies using an assay based on two I-SceI sites (22), whereas other studies have suggested that loss of these proteins has a significant effect on repair rates in similarly designed NHEJ assays (24,28). Finally, DSB repair assays recently have been described in which mutagenic NHEJ repair of a nuclease-induced DSB causes loss of fluorescent protein expression via disruption of the ORF, or a specific insertion/deletion restores the ORF of a frame-shifted fluorescent protein (29–31). These findings suggest that additional assays to measure NHEJ repair are needed to further elucidate these sub-pathways and also the roles of individual DSB repair proteins.

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