The possible involvement of DNA-dependent protein kinase in the phosphorylation of P1 protein in cell-free DNA replication of 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 P1 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.
Someya
A
A
Medical Information Center, Banyu Pharmaceutical Co., LTD, Tokyo, Japan.
Take
Y
Y
Shioda
M
M
D000595:Amino Acid Sequence; D000818:Animals; D034961:Antigens, Nuclear; D015153:Blotting, Western; D002474:Cell-Free System; D004265:DNA Helicases; D004261:DNA Replication; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000378:metabolism; D015536:Down-Regulation; D004789:Enzyme Activation; D000072200:Ku Autoantigen; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D017830:Octoxynol; D010766:Phosphorylation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D014981:Xenopus
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D017830:Octoxynol; D051747:DNA-Activated Protein Kinase; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen