Direct stimulation of poly(ADP ribose) polymerase in permeabilized cells by double-stranded DNA oligomers.
Poly(ADP ribosyl)ation, a post-translational modification of nuclear proteins catalyzed by poly (ADP ribose) polymerase, is an immediate response of most eukaryotic cells to DNA strand breaks and has been implicated in DNA repair and other cellular phenomena associated with DNA strand breakage. Poly(ADP ribose) polymerase activity levels have been frequently assayed by incubating permeabilized cells with radioactively labeled NAD+ as substrate. In such assays enzyme activation has routinely been achieved indirectly by prior exposure of living cells to carcinogens or by adding DNase I to permeabilized cells, thereby introducing strand breaks in chromosomal DNA. Here we show that, as an alternative method, the direct activation of purified poly(ADP ribose) polymerase by double-stranded oligonucleotides (N. A. Berger and S. I. Petzold, 1985, Biochemistry 24, 4352-4355) can be adopted for permeabilized cell systems. The inclusion of a palindromic decameric deoxynucleotide in the reaction buffer stimulated the enzyme activity in permeabilized Molt-3 human lymphoma cells up to 30-fold (at 50 micrograms/ml [corrected] oligonucleotide concentration) in a concentration-dependent manner. The activating effect of oligonucleotides was also evident when ethanol-fixed HeLa cells were postincubated with NAD+ to allow poly(ADP ribose) synthesis to occur in situ, which was detected as specific anti-poly (ADP ribose) immunofluorescence. We conclude that double-stranded oligonucleotides can be conveniently used as chemically and stoichiometrically well-defined poly (ADP ribose) polymerase activators in permeabilized or ethanol-fixed mammalian cells.
Grube
K
K
Institut für Virusforschung/ATV, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Küpper
J H
JH
Bürkle
A
A
D001483:Base Sequence; D004247:DNA; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D010539:Permeability; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D014407:Tumor Cells, Cultured / Q000201:enzymology
D009841:Oligonucleotides; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases