WT ATRIP cDNA but not cDNA encoding p.Arg760* ATRIP complements the G2/M checkpoint defect in CV1720 cells, and p.Arg760*ATRIP impairs ATR–ATRIP protein interaction.
A) Analysis of the G2/M checkpoint defect in CV1720 cells following expression of ATRIP cDNA. G2/M checkpoint arrest was examined 2 h post exposure to 5 Jm−2 UV. As shown in Figure 1A, WT cells showed proficient checkpoint arrest whilst DK0064 (ATR–SS) and CV1720 (patient) cells are unable to undergo arrest. Expression of WT ATRIP cDNA restored the ability of CV1720 (patient) and DK0064 (ATR–SS) to undergo checkpoint arrest but this was not observed following transfection of cDNA encoding R760* ATRIP. Significantly, expression of ATRIP R760* did not impair checkpoint arrest in WT cells verifying that it does not exert a dominant negative impact. Results represent the mean and SD of three experiments. WT cells were GM2188. ATR–SS represents DK0064 and patient, CV1720. B) R760* ATRIP impairs ATR–ATRIP interaction. Crude lysates were prepared from HEK293T cells and either mock transfected (lane1), transfected with HA-tagged WT ATRIP cDNA (lane2), or R760* ATRIP cDNA (lane3) (generating p.Arg760* ATRIP protein) together with ATR cDNA. The extracts were immunoprecipitated with agarose-conjugated rabbit anti-HA-tag antibody (MBL). Interaction with ATR was examined by immunoblotting with ATR antibodies (left panel). Immunoblotting using the HA-tag (ATRIP; right panel) verified expression of the appropriately sized ATRIP in the samples. 33% of the crude lysate was loaded; IP, immunoprecipitate.
