FIG. 2.

V3 transfectants expressing DNA-PKcs with multiple alanine but not aspartic acid substitutions within the PQR cluster are modestly radiosensitive. (A) Immunoblot analyses of whole-cell extracts (WCE) (50 μg) from V3 cell transfectants as indicated. (B) Radioresistance of V3 transfectants expressing wild-type DNA-PKcs, vector alone, or mutants P>ala, QR>ala, PQR>ala, or ABCDE>ala was assessed. In this figure and subsequent figures, data are presented as percent survival of unirradiated controls. Error bars indicate standard errors of the means for three independent experiments. (C) WCE prepared from V3 transfectants as indicated were assayed for DNA-PK activity. Each cell extract was tested in duplicate, and three independent extracts were tested. Kinase activity is expressed as [γ-³²P]ATP incorporation into the p53 target peptide divided by [γ-³²P]ATP incorporation in reactions without peptide, as described in reference 9. (D) Immunoblot analyses of WCE (20 μg) from V3 cells transfected with vector alone or wild-type DNA-PKcs expression vector or (200 μg) WCE from transfectants expressing mutants PQR>asp, P>asp, and QR>asp, as indicated. (E) Radioresistance of V3 transfectants expressing wild-type DNA-PKcs, vector alone, or mutant P>asp, QR>asp, PQR>asp, or PQR>ala was assessed. wt, wild type; vect., vector.