Adverse effect of MSH5 p.D487Y on DNA repair for DSBs
DSBs were induced by ETO treatment, and γH2AX foci were observed at the DSBs sites, which gradually disappeared along with the repair processing (Fig. 3A). The result showed that γH2AX level was much higher in the U2OS cells overexpressing mutant MSH5 than wild type (Fig. 3B). Furthermore, HeLa cells overexpressing mutant MSH5 showed a lower clonogenic survival rate than wild type, indicating the adverse effect of p.D487Y on DNA repair capacity or cellular resistance for DSBs. (Fig. 3C and D).
Figure 3 MSH5 p.D487Y impaired DNA repair. (A) Immunofluorescence showed the γH2AX foci formation in U2OS cells overexpressing wild type (WT) or mutant (D487Y) MSH5-GFP protein when suffering from ETO treatment. Scale bars: 5μm. (B) The γH2AX concentration among U2OS cells overexpressing blank vector (Control), wild type (WT) or mutant (D487Y) MSH5-Flag protein was detected by western blot. The expression of wild type and mutant MSH5 was detected by Flag antibody and β-actin was used as the loading control. (C) and (D) showed the clonogenic survival rate of WT- and D487Y-overexpressing cells in response to ETO treatment. The experiments on U2OS and HeLa cells both had 3 replicates independently. Data in the figure are shown as mean ± SD. M, siRNA targeting at MSH5; NT, non-targeting siRNA; and WT, wild type.