Figure 4.

G4122E mutant DNA-PKcs possesses weak DNA-PK activity. (A) Immunoblot analyses of whole cell extracts from V3 cells stably transfected with the following: wild-type human DNA-PKcs expression vector (lane 1); vector alone (lane 2); G4122E mutant DNA-PKcs expression vector (lanes 3); XR-C2 (lane 4). (B) Whole cell extracts (250 µg) prepared from V3 transfectants wild-type DNA-PKcs (black bar), pc-3 vector only (stippled bar), G4122E mutant protein (white bar) or XR-C2 (striped bar) were assayed for DNA-PK activity. Reactions were allowed to proceed for 10, 30 or 120 min as indicated. Each cell extract was tested in duplicate, and three independent extracts were tested for each cell line. Bars indicate the SD. (C) DNA cellulose fractions of 250 µg whole cell extracts from the following V3 transfectants: empty vector (lane 1); wild-type DNA-PKcs (lane 2); and G4122E mutant (lane 3) were incubated in kinase buffer with ³²P-labeled ATP. Subsequently, bound proteins were analyzed by silver staining of a 6% SDS–PAGE gel (left) and autoradiography (middle and right). (D) Radiation resistance of V3 transfectants expressing wild-type DNA-PKcs; vector only, G4122E mutant protein was compared to that of the CHOK9 and XR-C2 cell lines. Data is presented as percent survival of unirradiated controls and is a representative experiment from three different assays.