Figure 1. Mutation of S1333 to alanine creates a hyperactive ATR kinase.

(A–C) The indicated ATR mutant or wild type proteins complexed with ATRIP were purified from HEK293T cells and incubated with GST-MCM2 substrate, [γ-³²P]-ATP, and GST-TOPBP1-AAD (GST-AAD) or GST. Kinase reactions were separated by SDS-PAGE and [³²P]-MCM2 and [³²P]-GST-TOPBP1 detected by autoradiography. Quantitation of the fold activation compared to the wild type protein incubated with GST was measured by a phosphorimager and is indicated below each lane. The amount of ATR, TOPBP1, MCM2, and GST proteins in each reaction was detected with Coomassie blue (CB) or immunoblotting (IB) as indicated. Each mutant was tested at least three times and representative experiments are shown.