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Conditional deletion of Nbs1 in murine cells reveals its role in branching repair pathways of DNA double-strand breaks - PubMed

  • ️Sun Jan 01 2006

Conditional deletion of Nbs1 in murine cells reveals its role in branching repair pathways of DNA double-strand breaks

Yun-Gui Yang et al. EMBO J. 2006.

Abstract

NBS1 forms a complex with MRE11 and RAD50 (MRN) that is proposed to act on the upstream of two repair pathways of DNA double-strand break (DSB), homologous repair (HR) and non-homologous end joining (NHEJ). However, the function of Nbs1 in these processes has not fully been elucidated in mammals due to the lethal phenotype of cells and mice lacking Nbs1. Here, we have constructed mouse Nbs1-null embryonic fibroblasts and embryonic stem cells, through the Cre-loxP and sequential gene targeting techniques. We show that cells lacking Nbs1 display reduced HR of the single DSB in chromosomally integrated substrate, affecting both homology-directed repair (HDR) and single-stranded annealing pathways, and, surprisingly, increased NHEJ-mediated sequence deletion. Moreover, focus formation at DSBs and chromatin recruitment of the Nbs1 partners Rad50 and Mre11 as well as Rad51 and Brca1 are attenuated in these cells, whereas the NHEJ molecule Ku70 binding to chromatin is not affected. These data provide a novel insight into the function of MRN in the branching of DSB repair pathways.

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Figures

Figure 1
Figure 1

Generation and characterization of inducible Nbs1 deletion in mouse 3T3 MEFs. (A) Protocol used for generation of ‘inducible' Nbs1-null MEFs. (B) Southern blot, RT–PCR and Western blot analysis of Nbs1 deletion after OHT treatment in ‘inducible' Nbs1-null CER MEFs. The ‘f6' and ‘Δ6' alleles are indicated in the Southern blot. The genotype of each sample after OHT treatment is indicated at the bottom of the gels. Hprt served as an internal control for RT–PCR analysis. Actin is the loading control for Western blotting. (C) Colony formation assay of Nbs1 deletion in MEFs. Representative plates (upper panel) and quantification (lower panel) of colony formation assay are shown. Bars represent the mean of six clones of each sample from two independent experiments. (D) The proliferation curve of Nbs1-proficient (PSG±OHT and CER−OHT, f6/f6) and null (CER+OHT, Δ6/Δ6) MEFs. Curves represent the mean of duplicate samples from two independent experiments. (E) TUNEL analysis of inducible Nbs1-null MEFs (Δ6/Δ6) after treatment with adriamycin (+ADR) or not (−ADR) for 3 h (Time 0). TUNEL staining was performed at the indicated time points after drug removal. (F) Cytogenetic analysis of Nbs1f6/f6 or Nbs1neo/f6 CER cells after OHT-mediated Nbs1 deletion. Forty metaphases of each cell line were analyzed. (G) Spontaneous and ADR-induced micronucleus formation in the absence of Nbs1. Bars represent the mean of three clones of each genotype from two experiments.

Figure 2
Figure 2

Nbs1 depletion compromised both HDR and SSA in inducible Nbs1-null MEFs. (A) The HDR assay substrate DRGFP. (B) Nbs1-proficient or -null cells carrying a single intact copy of substrate DRGFP were analyzed by flow cytometry. Upper panel: representative flow cytometric analysis for HDR in MEFs after I-SceI expression. The GFP-positive population, reflecting HDR repair events, is separated from GFP-negative population. FL1, green fluorescence; FL2, orange fluorescence. Lower panel: The HDR events were scored as the percentage of GFP-positive populations after I-SceI expression. The percentage of GFP-positive cells in inducible (Δ6/Δ6) Nbs1-null mutants was set as 1. Bars represent the mean of seven MEF clones of each genotype from two independent experiments. (C) Strategy for analysis of SSA products in the DRGFP substrate. Four putative products surrounding the original genomic region of the I-SceI site: HDR, SSA, NHEJ and uncut DRGFP are shown. SSA was quantified by the products from primers DF and SAR, compared to that of the total PCR products from primers DF and DR (including HDR, SSA, DRGFP and NHEJ products). (D) Representative PCR-Southern blotting image (left panel) and quantification of SSA products (right panel) after I-SceI expression. The density of SSA products (DF-SAR) was normalized to the total PCR products (DF-DR) within the same sample. The SSA value of inducible (Δ6/Δ6) Nbs1-null mutants was set as 1. Bars represent the mean of three MEF clones of each genotype from two independent experiments. (E) SSA assay substrate SAGFP was stably integrated into MEFs and SSA was analyzed by flow cytometry after I-SceI transfection. (F) Summary of SSA in Nbs1-null (Δ6/Δ6) and -proficient (f6/f6) MEF cells. The SSA events were scored by the percentage of GFP-positive populations. The percentage of GFP-positive cells in Nbs1-null mutants was set as 1. Bars represent the mean of seven MEF clones of each genotype from two independent experiments. P-values by t-test are indicated in (B, D, F).

Figure 3
Figure 3

Increased NHEJ in MEF cells after induction of Nbs1 deletion. (A) PCR-Southern blot analysis of NHEJ. Representative gel (left panel) and quantification of NHEJ deletion products (right panel) after I-SceI expression. The PCR primers are shown in Figure 2C. The NHEJ frequency was examined by normalizing the density of NHEJ deletion products (Δ10–25 bp) to that of the 723-bp PCR product within the same sample. The NHEJ value of Nbs1-proficient cells (f6/f6) was set as 1. (B) Southern blot strategy for NHEJ analysis. NHEJ products are resistant to double digestion with I-SceI and BcgI enzymes. Note: the first HindIII site (star) is located outside the DRGFP substrate. Black filled box, I-SceI site arisen from uncut DRGFP; striped box, BcgI site retrieved from HDR and SSA products; empty box, NHEJ products resistant to both I-SceI and BcgI digestion. (C) Southern blot analysis of NHEJ using the strategy shown in (B). Representative Southern blot image (left panel) and quantification of total NHEJ products (right panel) in the DRGFP substrate after I-SceI expression. The NHEJ efficiency was examined by normalizing the density of NHEJ products (resistant to both I-SceI and BcgI) to that of cleaved product (0.9 kb, sensitive to both I-SceI and BcgI) within the same sample. The NHEJ value of Nbs1-proficient cells (f6/f6) was set as 1. Bars in (A, C) represent the mean of three MEF clones of each genotype from two independent experiments. P-values by t-test in (A, C) are indicated.

Figure 4
Figure 4

Generation and characterization of mouse ES cells and MEFs carrying constitutive Nbs1 deletion. (A) Protocol used to establish constitutive Nbs1-deleted MEFs from chimeric fetuses. (B) Western blot analysis of Nbs1-null ES cells. PARP-1 is a loading control. (C) Western blot analysis of Nbs1-null ES cells and 3T3 MEFs. Actin serves as a loading control. (D) Proliferation curve of constitutive Nbs1-null MEF cells. Curves represent the mean of triplicate samples from two independent experiments. (E) FACs analysis of cell cycle distribution of Nbs1-null ES cells. (F) Colony formation assay was performed to examine viability of constitutive Nbs1 deleted MEFs after ADR (0.2 μg/ml) or HU (2 mM) treatment. (G) Histogram shows the percentage of ES cells in sub-G1 phase extracted from FACs analysis at the indicated time points after 10 Gy of IR. Representative images of FISH analysis (H) and quantification (I) of wild type (+/+) and Nbs1-null (neo/hyg) ES cells subjected or not to 10 Gy of IR. Br: breaks; Fu: fusions. Between 16 and 19 metaphases from each treatment were analyzed and the results are shown as the mean±standard deviation. (J) SCE analysis of constitutive Nbs1-null MEFs with or without MMC treatment. At least 31 metaphases of each sample were scored and the t-test was applied for statistical analysis.

Figure 5
Figure 5

DSB repair defects in constitutive Nbs1-null ES cells. (A) The HDR events after I-SceI expression were analyzed by flow cytometry (see Figure 2B) and scored as the percentage of GFP-positive populations. The percentage of GFP-positive cells in Nbs1-null (neo/hyg) mutants was set as 1. Bars represent the mean of six ES clones of each genotype from two independent experiments. (B) Representative PCR-Southern blot image (left panel) and quantification of SSA products (right panel) in the HDR substrate DRGFP. The density of SSA products (DF-SAR) was normalized to the total PCR products (DF-DR) within the same sample. The SSA value of constitutive Nbs1-deleted mutants (neo/hyg) was set as 1. Bars represent the mean of three ES clones of each genotype from two independent experiments. (C) SSA analysis of Nbs1-null (neo/hyg) and -proficient (+/+) ES cells (see Figure 2E). The percentage of GFP-positive cells (SSA events) in Nbs1-null mutants was set as 1. Bars represent the mean of six ES clones of each genotype from two independent experiments. (D) Western blot analysis of transient ectopic expression of mouse Nbs1 cDNA in constitutive Nbs1-null ES cells harboring DRGFP. Defects in HDR (E) and SSA (F) are rescued by transient ectopic expression of mouse Nbs1 cDNA. One of two independent experiments are shown for (E) and (F). Fanconi anemia-A mutant fibroblasts (Fanca−/−; Yang et al, 2005) were used as a negative control. (G) PCR-Southern blot analysis of NHEJ activity in proliferating Nbs1-null ES cells as described in Figure 2C. Representative Southern blot image (left panel) and quantification of NHEJ deletion products (right panel). The NHEJ value of Nbs1-proficient cells (+/+) was set as 1. (H) Southern blot strategy for NHEJ analysis. Note: NHEJ products resistant to both I-SceI and BcgI digestions are ⩾4.8 kb. (I) Representative Southern blotting image (left panel) and quantification of total NHEJ products (right panel). The NHEJ efficiency was examined as described in Figure 3C. The NHEJ value of Nbs1-proficient cells (+/+) was set as 1. Bars in (G, I) represent the mean of three ES clones of each genotype from two independent experiments. The t-test was applied for statistical analysis.

Figure 6
Figure 6

Defective focus formation of DSB repair molecules in MEF cells lacking Nbs1. Immunofluorescence analysis of Rad51 (A) and Brca1 (B) focus formation in inducible (Δ6/Δ6) and constitutive (neo/hyg) Nbs1-null MEFs after treatment with 0.2 μg/ml ADR for 3 h. Lower panels of (A) and (B) are quantifications of DNA damage-induced foci. Bars represent means±s.d. are shown for duplicate samples from two independent experiments. For each sample at least 500 nuclei were scored. (C) Western blot analysis of the chromatin-enriched fraction of MRN and Ku70 proteins. 0 h, 0 h after removal of ADR; 6 h, 6 h after removal of ADR. PARP-1 is served as a control.

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