Fig. 5: RAD18 O-GlcNAcylation is essential for its binding with both ubiquitin and RAD51C.

A RAD18 O-GlcNAcylation promotes HR repair. DR-GFP U2OS cells overexpressing Mcherry empty vector, WT or 3A Mcherry-Flag-RAD18 were treated with siRAD18 oligo followed by I-SceI endonuclease transfection. HR repair efficiency (GFP⁺Mcherry⁺%) was analyzed by FACS. The lower panels show immunoblots indicating the RAD18 levels in different conditions. B RAD18 O-GlcNAcylation promotes cell survival post-CPT treatment. Clonogenic survival assays in RAD18-knockdown U2OS cells transfected with empty vector, WT or 3A Flag-RAD18 after CPT treatment. Cells were treated with indicated doses of CPT and further incubated for 7–10 days. Surviving fraction was expressed as a percentage of mock-treated cells. The representative curve is shown. Error bar: s.d., n = 3. C Representative images for colony assay in (B). D 3A mutation inhibits the binding ability of RAD18 with ubiquitin. Purified GST and GST-Ubb were incubated with HEK293T cell lysates expressing SFB-RAD18 (WT or 3A) followed by immunoblotting with Flag antibody. Ponceau staining shows the amounts of GST and GST-Ubb used in the pulldown assay. E RAD18-depleted HEK293T cells were transfected with WT or 3A GFP-RAD18 and SFB-RAD51C. The cell lysates were denatured followed by immunoprecipitation with anti-GFP agarose. The immunoprecipitates were immunoblotted with Flag and GFP antibodies. F Purified His-RAD51C was incubated with OGT knockdown HEK293T cell lysates expressing SFB-RAD18 in incubation with glucose (60 mM) and TMG (5 μM) or not. The bound proteins were analyzed as in (D).