Figure 4.

Merit40 mutation reduces ICL unhooking efficiency and HR repair of ICL damage. (A,B) Representative images and quantification of RAP80 and FANCD2 at laser-induced psoralen cross-links in HeLa cells. The arrows indicate positive recruitment signals. (C) Cells were treated for 1 h with 50 μM cisplatin followed by cisplatin removal. Samples were taken at different time points after cisplatin treatment, and unhooking of ICL was measured using a modified comet assay (please see the Materials and Methods for a detailed description). Immediately before analysis, cells collected from different time points were exposed to 12.5 Gy of IR. DNA interstrand cross-linking was expressed as percentage decrease in tail moment compared with irradiated controls calculated by the following formula: percent decrease in Olive tail moment = [1 − (TMdi − TMcu)/(TMci − TMcu)] × 100, where TMdi is the tail moment of the cisplatin-treated, irradiated sample; TMcu is the tail moment of the untreated, unirradiated control; and TMci is the tail moment of the untreated, irradiated control. (D) MEFs were labeled with BrdU for two cell cycles and then treated with 20 ng/mL MMC for the last 24 h prior to harvesting for metaphase analysis of sister chromatid exchanges. Quantification was derived from four independent experiments. (E–G) HeLa cells depleted of MERIT40 by three individual siRNAs show reduced recruitment of BRCA1 (E), Rad51 (F), and RPA (G) to psoralen-induced ICLs. The mean intensities of protein colocalized to ICL stripes were quantified in single cells at 20 min after laser treatment.