Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam 13620, Korea.
Department of Internal Medicine, Seoul National University College of Medicine, Seoul 03080, Korea.
Abstract
Repairing damaged DNA and removing all physical connections between sister chromosomes is important to ensure proper chromosomal segregation by contributing to chromosomal stability. Here, we show that the depletion of non-SMC condensin I complex subunit H (NCAPH) exacerbates chromosome segregation errors and cytokinesis failure owing to sister-chromatid intertwinement, which is distinct from the ultra-fine DNA bridges induced by DNA inter-strand crosslinks (DNA-ICLs). Importantly, we identified an interaction between NCAPH and GEN1 in the chromatin involving binding at the N-terminus of NCAPH. DNA-ICL activation, using ICL-inducing agents, increased the expression and interaction between NCAPH and GEN1 in the soluble nuclear and chromatin, indicating that the NCAPH-GEN1 interaction participates in repairing DNA damage. Moreover, NCAPH stabilizes GEN1 within chromatin at the G2/M-phase and is associated with DNA-ICL-induced damage repair. Therefore, NCAPH resolves DNA-ICL-induced ultra-fine DNA bridges by stabilizing GEN1 and ensures proper chromosome separation and chromosome structural stability.
Keywords
DNA inter-strand crosslinkDNA repairGEN1NCAPHchromosomal stabilitycondensin
Figures
Fig. 1 Response of NCAPH by DNA-ICL induction using MMC and cisplatin treatment. (A) Introduction to a brief overview of the procedure for double thymidine block synchronization. (B) MIA PaCa-2 cells were treated with 2 μM MMC from the second thymidine treatment, and then analyzed at separate time points ranged from 0, 4, and 8 h after release of second thymidine treatment. Comparison of the chromatin– and chromatin+ fractions of MIA PaCa-2 cells. Western blotting analysis was performed to show protein expression levels of NCAPH, p-H2AX, and LaminA/C (LMNA); nuclear marker. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH); cytoplasmic marker. (C) MIA PaCa-2 cells were treated with 2 μM MMC in the second thymidine treatment and then subjected to immunofluorescence analysis at 8 h after release from the second thymidine treatment. p-H2AX (green) and NCAPH (red) fluorescence pattern was observed by confocal microscopy. DNA was stained with DAPI (blue). Scale bars = 5 μm. (i and ii) Colocalization analysis of NCAPH (red curve) and p-H2AX (green curve) with DAPI (blue curve). The fluorescence intensity profiles of NCAPH and p-H2AX show the fluorescence distribution across the dotted line (x-axis). The fluorescence intensity is plotted along the y-axis. (D) Measurements of the mean fluorescence intensity of NCAPH. For precise quantification, more than 100 cell images captured by fluorescence microscopy at least three different fields. Values are represented in the form of mean ± SEM. Statistical analysis of data was obtained by unpaired t-tests. ***P < 0.0001. (E) MIA PaCa-2 cells were transfected with control and NCAPH siRNA, and treated with DMSO, 1 μg/ml cisplatin, or 2 μM MMC for 24 h. Analyze the cell cycle by flow cytometry. (F) Histogram bar graph visualize the percentages in cells in sub-G1 (red), G1 (blue), S (orange), and G2/M (green) phases. The experiments were performed in triplicate, repeated at least three times. The mean values of the results are presented. *P < 0.005, two-way ANOVA. (G) MIA PaCa-2 cells were transfected with control and NCAPH siRNA, and treated with DMSO, 1 μg/ml cisplatin, or 2 μM MMC for 24 h. The protein expression levels of NCAPH, p-H2AX, cyclin A2, and β-actin were measured by western blotting.Fig. 2 NCAPH-depleted cells induce UFB formation, chromosome segregation errors, and cytokinesis failure by cisplatin-induced DNA-ICLs. (A) To confirm the formation of UFBs, HEK293 cells were transfected with control and NCAPH siRNA, and treated with 1 μg/ml cisplatin for 1 h. After 24 h, immunofluorescence was performed using anti-PICH. DNA was stained with DAPI (blue). Scale bars = 5 μm. For precise quantification, more than 300 cells captured images by fluorescence microscopy at least three independent experiments were analyzed. Values are represented in the form of mean ± SEM. Statistical analysis of data was obtained by unpaired t-tests. ***P < 0.0001. (B) HEK293 cells transfected with control or NCAPH siRNA were stained for CREST (green), α-tubulin (red), and DNA (blue). Scale bars = 5 μm. For precise quantification, more than 300 cells captured images by fluorescence microscopy at least three independent experiments were analyzed. Values are represented in the form of mean ± SEM. Statistical analysis of data was obtained by unpaired t-tests. ***P < 0.0001. (C) MIA PaCa-2 cells transfected with control or NCAPH siRNA were stained for DNA (gray). Scale bars = 10 μm. For precise quantification, more than 300 cells captured images by fluorescence microscopy at least three independent experiments were analyzed. Values are represented in the form of mean ± SEM. ***P < 0.0001; ns, not significant. Statistical analysis of data was obtained by one-way ANOVA followed by Bonferroni’s multiple comparison analysis tests.Fig. 3 NCAPH interacts with GEN1 to mediated DNA repair of MMC-induced DNA-ICLs. (A) HEK293 cells were transfected Flag-tagged full-length NCAPH or control vector. Total lysates in input and immunoprecipitated (IP) with anti-Flag M2 affinity gel, and then analyzed by immunoblotting (IB) with specific antibodies (GEN1, MUS81, NCAPH, and NCAPD2 antibodies). (B) HEK293 cells were transfected Flag-tagged full-length GEN1 or control vector. Total lysates in input and immunoprecipitated with anti-Flag M2 affinity gel, and then analyzed by immunoblotting with specific antibodies (NCAPH and Flag antibodies). (C) HEK293 cells were transfected Flag-tagged full-length NCAPH and GST-tagged full-length GEN1. Total lysates in input and immunoprecipitated with anti-Flag M2 affinity gel or Glutathione-Sepharose beads, and then analyzed by immunoblotting with specific antibodies (GST and Flag antibodies). (D) HEK293 cells were transfected Flag-tagged full-length NCAPH and GST-tagged full-length GEN1 with ATM-kinase inhibitor treatment for 24 h, and immunoprecipitation analysis. Comparison of the cytoplasmic and chromatin fractions of HEK293 cells prepared by lysis through chromatin fractionation. Total lysates in input and immunoprecipitated with Glutathione-Sepharose beads, and then analyzed by immunoblotting with specific antibodies (Flag, GST, GAPDH, and LMNA antibodies). (E) Diagram of full-length NCAPH and three truncation domain constructs (N-NCAPH [N], M-NCAPH [M], C-NCAPH [C]). Protein size is given in amino acids (aa). (F) HEK293 cells were transfected three domains of NCAPH and GST-tagged full-length GEN1. Total lysates in input and immunoprecipitated with anti-Flag M2 affinity gel or Glutathione-Sepharose beads, and then analyzed by immunoblotting with specific antibodies (Flag, GST, and NCAPD2 antibodies). (G) HEK293 cells were transfected Flag-tagged full-length NCAPH and GST-tagged full-length GEN1 and then treated with 2 μM MMC in the second thymidine treatment. Immunoprecipitation assay was performed at 0, 4, 8 h after release from the second thymidine treatment. Comparison of the cytoplasmic and chromatin fractions of HEK293 cells prepared by lysis through chromatin fractionation. Total lysates in input and immunoprecipitated with Glutathione-Sepharose beads, and then analyzed by immunoblotting with specific antibodies (Flag, GST, GAPDH, and LMNA antibodies). Flag-NCAPH, Flag-tagged full-length NCAPH; Flag-GEN1, Flag-tagged full-length GEN1; GST-GEN1, GST-tagged full-length GEN1.Fig. 4 NCAPH-depleted cells dramatically reduced GEN1 recruitment and stabilization in chromatin at G2/M-phase. (A) Introduction to a brief overview of the procedure for double thymidine block synchronization. (B) MIA PaCa-2 cells were transfected with control and NCAPH siRNA and double thymidine block synchronization was performed. The cells were analyzed for 0, 4, 8, and 12 h after release from the second thymidine treatment. Analyze the cell cycle by flow cytometry. Line graph visualize the percentages in cells in S and G2/M-phase population. Values represent mean ± SEM. *P < 0.005, **P < 0.001. Results were analyzed using unpaired t-tests. (C) The protein expression levels of NCAPH, cyclin A2, pS10 histone H3, and β-actin were measured by western blotting. (D) MIA PaCa-2 cells were transfected with control and NCAPH siRNA and double thymidine block synchronization was performed. The cells were analyzed for 0 and 8 h after release from the second thymidine treatment. Comparison of the Cyt (cytoplasmic), Nuc (soluble nuclear), and Chr (chromatin) fractions of MIA PaCa-2 cells prepared by lysis through chromatin fractionation experiments. Western blotting analysis was performed to show protein expression levels of NCAPH, NCAPG, NCAPH2, NCAPD3, GEN1, α-tubulin, GAPDH, LMNA, Histone H3. (E) MIA PaCa-2 cells were treated with 2 μM MMC in the second thymidine treatment and then subjected to immunofluorescence analysis at 8 h after release from the second thymidine treatment p-H2AX (green) and GEN1 (red) fluorescence pattern was observed by confocal microscopy. DNA was stained with DAPI (blue). Scale bars = 5 μm. (i and ii) Colocalization analysis of NCAPH (red curve) and p-H2AX (green curve) with DAPI (blue curve). The fluorescence intensity profiles of NCAPH and p-H2AX show the fluorescence distribution across the dotted line (x-axis). The fluorescence intensity is plotted along the y-axis.Fig. 5 NCAPH required for GEN1 recruitment and stabilization to chromatin. (A) Western blotting analysis of total cell lysates harvested after control and NCAPH siRNA transfection into MIA PaCa-2 cells and dose-dependent treatment of Lenti-siR-NCAPH. Western blotting analysis was performed to show protein expression levels of NCAPH and β-actin. (B) Comparison of the Cyt (cytoplasmic), Nuc (soluble nuclear), and Chr (chromatin) fractions of MIA PaCa-2 cells prepared by lysis through chromatin fractionation experiments. Western blotting analysis was performed to show protein expression levels of NCAPH, GEN1, GAPDH, and LMNA. (C) MIA PaCa-2 cells were treated with or without Lenti-siR-NCAPH after transfection with control and NCAPH siRNA. After 48 h, p-H2AX (green) and GEN1 (red) fluorescence patterns was observed by confocal microscopy. DNA was stained with DAPI (blue). Scale bars = 5 μm. (i-iii) Colocalization analysis of NCAPH (red curve) and p-H2AX (green curve) with DAPI (blue curve). The fluorescence intensity profiles of NCAPH and p-H2AX show the fluorescence distribution across the dotted line (x-axis). Fluorescence intensity is plotted along the y-axis. (D) Calculation of colocalization of GEN1 and p-H2AX fluorescence. For precise quantification, more than 40 cells captured images by fluorescence microscopy at least three different fields were analyzed using the Pearson correlation coefficient (PCC) and the Mander’s overlap coefficient (MOC) measurement methods. Values are represented in the form of mean ± SEM. ***P < 0.0001. Statistical analysis of data was obtained by one-way ANOVA followed by Bonferroni’s multiple comparison analysis tests.
그림 출처: PubMed Central 공개 논문 (PMC9676985)
Conflict of interest statement
CONFLICT OF INTEREST . The authors have no potential conflicts of interest to disclose.
