CSA and CSB play a role in the response to DNA breaks

Abstract

CS proteins have been involved in the repair of a wide variety of DNA lesions. Here, we analyse the role of CS proteins in DNA break repair by studying histone H2AX phosphorylation in different cell cycle phases and DNA break repair by comet assay in CS-A and CS-B primary and transformed cells. Following methyl methane sulphate treatment a significant accumulation of unrepaired single strand breaks was detected in CS cells as compared to normal cells, leading to accumulation of double strand breaks in S and G2 phases. A delay in DSBs repair and accumulation in S and G2 phases were also observed following IR exposure. These data confirm the role of CSB in the suppression of NHEJ in S and G2 phase cells and extend this function to CSA. However, the repair kinetics of double strand breaks showed unique features for CS-A and CS-B cells suggesting that these proteins may act at different times along DNA break repair. The involvement of CS proteins in the repair of DNA breaks may play an important role in the clinical features of CS patients.

Keywords: Cockayne syndrome; DNA damage; DNA repair; γ-H2AX.

Figure 1. Evaluation of SSBs formation and repair in CS-A and CS-B cells after MMS exposure

Normal and CS primary fibroblasts were exposed to 0.5 mM MMS for 30 min and repair kinetics was followed for different times. SSBs were measured by alkaline SCGE. Normal: N1RO and N2RO; CS-A: CS6PV and CS7PV; CS-B: CS20PV and CS1AN. (A) Microphotographs of normal, CS-A and CS-B cells, without treatment (control), immediately after treatment (0.5 mM) and after different repair times (1 h and 3 h). (B) Data from three independent experiments were reported as box plots. Each box encloses 50% of the data. The median of the distribution, the acceptable range and outliers are indicated. (C) Normal and CS primary fibroblasts synchronized in G1 phase were exposed to 0.5 mM MMS for 30 min and SSB were measured by alkaline SCGE and the repair kinetics was followed for different times (30 min and 2 h). Box plot shows data presented as tail moment. Box and whiskers represent 25–75 and 10–90 percentiles, respectively. The line represents the median value. Means of three independent experiments ± SE are reported; ^*p < 0.01, ^**p < 0.001 by nonparametric Wilcoxon ranksum test. Normal: N2RO; CS-A: CS6PV; CS-B: CS20PV.

Figure 2. Flow cytometry analysis of histone H2AX phosphorylation in CS-A and CS-B cells in the different phases of the cell cycle after MMS treatment

Normal, CS-A and CS-B primary fibroblasts were double stained with PI and an anti γ-H2AX antibody. The mean γ-H2AX fluorescence of G1-, S- and G2/M-phase selected cells of both untreated (control) and 0.5 mM MMS treated is shown. Normal: N2RO; CS-A: CS24PV; CS-B: CS20PV. Means of three independent experiments ± SE are reported. The mean fluorescence of γ-H2AX is expressed in arbitrary units (AU).

Figure 3. Evaluation of DSB formation and repair in CS-A and CS-B cells after MMS exposure

Cells were treated with 0.5 mM MMS for 30 min and repair kinetics was followed for different times (1 h-6 h). DSBs were measured by neutral SCGE on CS-A (CS3BE) (A) and CS-B (SV40-CS1AN) cells (B) Box plot shows data presented as tail moment. Box and whiskers represent 25–75 and 10–90 percentiles, respectively. The line represents the median value. Means of three independent experiments are reported; ^**p < 0.001, ^***p < 0.0001, Kruskal-Wallis test multicomparison Anova.

Figure 4. Flow cytometry analysis of histone H2AX phosphorylation on CS-A and CS-B cells in the different phases of the cell cycle after IR

Normal, CS-A and CS-B primary fibroblasts were double stained with PI and an anti γ-H2AX antibody. The mean γ-H2AX fluorescence of G1-, S- and G2-phase selected cells of both untreated (control) and 3 Gy ionizing radiation treated is shown. Normal: N2RO; CS-A: CS24PV; CS-B: CS20PV. Means of three independent experiments ± SE are reported. The mean fluorescence of γ-H2AX is expressed in arbitrary units (AU).

Figure 5. Evaluation of DSB formation and repair in CS-A and CS-B cells after exposure to IR

Cells were treated with 10 Gy of ionizing radiation and repair kinetics was followed for different times (1 h-6 h). DSBs were measured by neutral SCGE on CS-A (CS3BE) (A) and CS-B (SV40-CS1AN) (B) transformed fibroblasts, and their isogenic derivatives expressing the wild-type genes (CS3BE-wtCS-A and SV40-CS1AN-wtCS-B). Box plot shows data presented as tail moment. Box and whiskers represent 25–75 and 10–90 percentiles, respectively. The line represents the median value. Means of three independent experiments are reported; ^**p < 0.001, ^***p < 0.0001, Kruskal-Wallis test multicomparison Anova.