THE DNA-DAMAGE RESPONSE IN DNA-DEPENDENT PROTEIN KINASE-DEFICIENT SCID MOUSE CELLS - REPLICATION PROTEIN-A HYPERPHOSPHORYLATION AND P53 INDUCTION

Severe combined immunodeficient (SCID) mice display an increased sensi tivity to ionizing radiation compared with the parental, C.B-17, strai n due to a deficiency in DNA double-strand break repair, The catalytic subunit of DNA-dependent protein kinase (DNA-PKCS) has previously bee n identified as a strong candidate for the SCID gene. DNA-Ps phosphory lates many proteins in vitro, including p53 and replication protein A (RPA), two proteins involved in the response of cells to DNA damage. T o determine whether p53 and RPA are also substrates of DNA-PK in vivo following DNA damage, we compared the response of SCID and MO59J (huma n DNA-PKcs-deficient glioblastoma) cells with their respective wild-ty pe parents following ionizing radiation, Our findings indicate that (i ) p53 levels are increased in SCID cells following ionizing radiation, and (ii) RPA p34 is hyperphosphorylated in both SCID cells and MO59J cells following ionizing radiation. The hyperphosphorylation of RPA p3 4 in vivo is concordant with a decrease in the binding of RPA to singl e-stranded DNA in crude extracts derived from both C.B-17 and SCID cel ls, These results suggest that DNA-PK is not the only kinase capable o f phosphorylating RPA, We conclude that the DNA damage response involv ing p53 and RPA is not associated with the defect in DNA repair in SCI D cells and that the physiological substrate(s) for DNA-PK essential f or DNA repair has not yet been identified.