DNA damage and recovery assay
The human osteosarcoma U2OS cells were transiently transfected with wild type or mutant MSH5-GFP or MSH5-Flag plasmids, and were incubated in media containing Etoposide (ETO) (5μg/ml) for 1 h at 37°C to induce DSBs, followed with normal media replacement and recovery for 2 h at 37°C. Phosphorylation of the Ser-139 residue of histone variant H2AX, forming γH2AX, is an early cellular response to DSBs, which here was used as a sensitive marker for DSBs. Immunofluorescence and western blot were performed to detect DNA damage and recovery degree by staining γH2AX. The clonogenic survival rate after ETO treatment was calculated in HeLa cells, in which endogenous MSH5 was silenced with siRNA and then wild type or mutant MSH5 was overexpressed.