Measuring WRN-dependent DNA repair efficiency

HEK293TshWRN cells were seeded in six-well plates at a density of 0.5 million cells per well. The next day, pCMV-FlagRnai-resWRN^WT, pCMV-FlagRnai-resWRN^S1133A or pCMV-FlagRnai-resWRN^S1133D were cotransfected with the I-SceI expression vector pCBASceI and the pHPRT-DRGFP plasmid reporter using DreamFect (OZ Biosciences) according to the manufacturer's instruction. Alternatively, the I-SceI linearized NHEJ reporter40 (link) replaced the pHPRT-DRGFP plasmid. pHPRT-DRGFP and pCBASceI were a gift from Maria Jasin (Addgene plasmids #26476 and #26477). Protein expression levels were analysed by western blotting 72 h post transfection. Cells were subjected to flow cytometry analysis at 72 h after transfection to determine the percentage of GFP-positive cells from 1 × 10⁵ events. After correction for background level of GFP-positive cells obtained from no-I-Sce-I transfected cells, results were reported as percentage of repair efficiency compared with the number of GFP-positive cells observed in the presence of wild-type WRN.