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A nonsense mutation of human XRCC4 is associated with adult-onset progressive encephalocardiomyopathy.

Bee L, Nasca A, Zanolini A, Cendron F, d'Adamo P, Costa R, Lamperti C, Celotti L, Ghezzi D, Zeviani M - EMBO Mol Med (2015)

Bottom Line: XRCC4 transcript levels were profoundly reduced, and the protein was undetectable in patients' skin fibroblasts.Gamma-irradiated mutant cells demonstrated reduction, but not abolition, of DSB repair.Surprisingly, neither immunodeficiency nor predisposition to malignancy was reported in these patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Padua, Padua, Italy.

No MeSH data available.


Related in: MedlinePlus

Analysis of PARP-1 inhibition on DSB repairXRCC4wt and XRCC4m/m cells irradiated with 0.5 Gy of γ-rays were exposed to the PARP1 inhibitor 3′-AB or to solvent (DMSO). The γ-H2AX foci number is the mean ± SD of three independent experiments. *P < 0.05 (XRCC4m/m + 3′-AB versus XRCC4m/m + DMSO), ***P < 0.001 (XRCC4m/m + 3′-AB versus XRCC4wt + 3′-AB), two-way ANOVA, Bonferroni post hoc test.
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fig05: Analysis of PARP-1 inhibition on DSB repairXRCC4wt and XRCC4m/m cells irradiated with 0.5 Gy of γ-rays were exposed to the PARP1 inhibitor 3′-AB or to solvent (DMSO). The γ-H2AX foci number is the mean ± SD of three independent experiments. *P < 0.05 (XRCC4m/m + 3′-AB versus XRCC4m/m + DMSO), ***P < 0.001 (XRCC4m/m + 3′-AB versus XRCC4wt + 3′-AB), two-way ANOVA, Bonferroni post hoc test.

Mentions: The fraction of cycling cells in a population of skin fibroblasts is usually low. In our experimental conditions (Table1), most of the cells were in G1 phase, as detected by analyzing DNA content. Before irradiation, 88.62 ± 1.86% of XRCC4wt fibroblasts was in G1 phase and 6.41 ± 0.35% in G2/M phases; 24 h after irradiation, G2 cells increased up to 11.09 ± 0.80%, probably because of G2-checkpoint activation. In XRCC4m/m non-irradiated cells, the percentages of G1 phase was 92.23 ± 0.43% and that of G2/M phases 5.25 ± 1.47%. At 24 h after irradiation, XRCC4m/m cells in G1 phase were 93.91 ± 0.33% and those in G2/M were unchanged (5.44 ± 0.77%), probably because of failure of G2 checkpoint activation. Thus, the increased recruitment of HR factors to DNA lesions detected in XRCC4m/m-mutant cells in G2 phase (RAD51 foci in Fig4), which was about 5% of the total, cannot explain the persistence of the DNA repair level measured in our experiments. We therefore analyzed the involvement of the alternative non-homologous end-joining (A-NHEJ) pathway in DSB repair. The nuclear enzyme PARP-1 is usually involved in SSB rejoining by BER or NER processes, but in NHEJ-defective cells, it has been shown to participate in DSB repair, as a component of A-NHEJ (Wang et al, 2006; Mansour et al, 2010; Mladenov & Iliakis, 2011). To calculate the residual DSB rejoining upon inhibition of the A-NHEJ system, we treated XRCC4wt and XRCC4m/m fibroblasts with a PARP-1 inhibitor, 3′-aminobenzamide (3′-AB), 24 h before irradiation (Fig5). From 2 h after irradiation, the number of γ-H2AX foci in 3′-AB treated XRCC4m/m cells was significantly higher than in untreated XRCC4m/m cells. Conversely, in XRCC4wt cells, where DSB repair is performed by the classical NHEJ (C-NHEJ) pathway, 3′-AB treatment did not modify the number of γ-H2AX foci found in untreated cells. We observed no difference in the amount of PARP-1 by Western blot analysis of XRCC4m/m vs. XRCC4wt cells, possibly because of qualitative changes in distribution (e.g. relocation of the protein within cell compartments) rather than quantitative variations in content.


A nonsense mutation of human XRCC4 is associated with adult-onset progressive encephalocardiomyopathy.

Bee L, Nasca A, Zanolini A, Cendron F, d'Adamo P, Costa R, Lamperti C, Celotti L, Ghezzi D, Zeviani M - EMBO Mol Med (2015)

Analysis of PARP-1 inhibition on DSB repairXRCC4wt and XRCC4m/m cells irradiated with 0.5 Gy of γ-rays were exposed to the PARP1 inhibitor 3′-AB or to solvent (DMSO). The γ-H2AX foci number is the mean ± SD of three independent experiments. *P < 0.05 (XRCC4m/m + 3′-AB versus XRCC4m/m + DMSO), ***P < 0.001 (XRCC4m/m + 3′-AB versus XRCC4wt + 3′-AB), two-way ANOVA, Bonferroni post hoc test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4520657&req=5

fig05: Analysis of PARP-1 inhibition on DSB repairXRCC4wt and XRCC4m/m cells irradiated with 0.5 Gy of γ-rays were exposed to the PARP1 inhibitor 3′-AB or to solvent (DMSO). The γ-H2AX foci number is the mean ± SD of three independent experiments. *P < 0.05 (XRCC4m/m + 3′-AB versus XRCC4m/m + DMSO), ***P < 0.001 (XRCC4m/m + 3′-AB versus XRCC4wt + 3′-AB), two-way ANOVA, Bonferroni post hoc test.
Mentions: The fraction of cycling cells in a population of skin fibroblasts is usually low. In our experimental conditions (Table1), most of the cells were in G1 phase, as detected by analyzing DNA content. Before irradiation, 88.62 ± 1.86% of XRCC4wt fibroblasts was in G1 phase and 6.41 ± 0.35% in G2/M phases; 24 h after irradiation, G2 cells increased up to 11.09 ± 0.80%, probably because of G2-checkpoint activation. In XRCC4m/m non-irradiated cells, the percentages of G1 phase was 92.23 ± 0.43% and that of G2/M phases 5.25 ± 1.47%. At 24 h after irradiation, XRCC4m/m cells in G1 phase were 93.91 ± 0.33% and those in G2/M were unchanged (5.44 ± 0.77%), probably because of failure of G2 checkpoint activation. Thus, the increased recruitment of HR factors to DNA lesions detected in XRCC4m/m-mutant cells in G2 phase (RAD51 foci in Fig4), which was about 5% of the total, cannot explain the persistence of the DNA repair level measured in our experiments. We therefore analyzed the involvement of the alternative non-homologous end-joining (A-NHEJ) pathway in DSB repair. The nuclear enzyme PARP-1 is usually involved in SSB rejoining by BER or NER processes, but in NHEJ-defective cells, it has been shown to participate in DSB repair, as a component of A-NHEJ (Wang et al, 2006; Mansour et al, 2010; Mladenov & Iliakis, 2011). To calculate the residual DSB rejoining upon inhibition of the A-NHEJ system, we treated XRCC4wt and XRCC4m/m fibroblasts with a PARP-1 inhibitor, 3′-aminobenzamide (3′-AB), 24 h before irradiation (Fig5). From 2 h after irradiation, the number of γ-H2AX foci in 3′-AB treated XRCC4m/m cells was significantly higher than in untreated XRCC4m/m cells. Conversely, in XRCC4wt cells, where DSB repair is performed by the classical NHEJ (C-NHEJ) pathway, 3′-AB treatment did not modify the number of γ-H2AX foci found in untreated cells. We observed no difference in the amount of PARP-1 by Western blot analysis of XRCC4m/m vs. XRCC4wt cells, possibly because of qualitative changes in distribution (e.g. relocation of the protein within cell compartments) rather than quantitative variations in content.

Bottom Line: XRCC4 transcript levels were profoundly reduced, and the protein was undetectable in patients' skin fibroblasts.Gamma-irradiated mutant cells demonstrated reduction, but not abolition, of DSB repair.Surprisingly, neither immunodeficiency nor predisposition to malignancy was reported in these patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Padua, Padua, Italy.

No MeSH data available.


Related in: MedlinePlus