THE POSSIBLE INVOLVEMENT OF DNA-DEPENDENT PROTEIN-KINASE IN THE PHOSPHORYLATION OF P1 PROTEIN IN CELL-FREE DNA-REPLICATION XENOPUS EGGS

We prepared two types of antibodies: one directed against an oligopeptide corresponding to the C-terminal portion of P1 protein and the other against an oligopeptide corresponding to the C-terminal portion of the 80-kDa subunit of the Ku antigen (p80 Ku) essential for DNA-dependent protein kinase (DNA-PK) activity. Immunoprecipitation and immunoblot of sperm nuclei preincubated in Xenopus egg extracts by anti-P1 antibody showed that Xenopus P1 protein is a phosphoprotein with two phosphorylated forms: a hyperphosphorylated form extractable with Triton X-100 and a hypophosphorylated form resistant to Triton X-100. The immunodepletion of extracts with anti-p80 Ku IgG-bound beads caused the hyperphosphorylated form to disappear but hardly affected the hypophosphorylated form of Fl protein. DNA replication was stimulated by immunodepletion of the extract with anti-p80 Ku IgG-bound beads. These findings suggest that DNA-PK down-regulates DNA replication through inhibition of hyperphosphorylation of P1 protein during S phase in this cell-free system. (C) 1995 Academic Press, Inc.