Inhibition of poly(ADP-ribosyl)ation by overexpressing the poly(ADP-ribose) polymerase DNA-binding domain in mammalian cells.
Poly(ADP-ribose) polymerase specifically recognizes DNA strand breaks by its DNA-binding domain. DNA binding activates the enzyme to catalyze the formation of poly(ADP-ribose) utilizing NAD as substrate. By a molecular genetic approach we set out to inhibit this enzyme activity in a highly specific manner, thus avoiding the inherent side effects of NAD analogs which have been used extensively as enzyme inhibitors. cDNA sequences coding for the human poly(ADP-ribose) polymerase DNA-binding domain were subcloned into eucaryotic expression plasmids and transiently transfected into monkey cells. Cells were fixed with ethanol followed by incubation with NAD. Indirect double immunofluorescence to detect both overexpressed protein and poly(ADP-ribose) in situ revealed that overexpression of the DNA-binding domain greatly inhibited poly(ADP-ribosyl)ation catalyzed by the resident enzyme during NAD postincubation. The same inhibition was observed when transfected cells were treated with N-methyl-N'-nitro-N-nitrosoguanidine to induce DNA strand breaks in vivo and subjected to trichloroacetic acid/ethanol fixation and subsequent immunofluorescence analysis, a novel method we developed for the in situ detection of polymer synthesis in intact cells. This molecular genetic approach may prove to be a selective and efficient tool to investigate possible functions of poly(ADP-ribosyl)ation in living cells.
Küpper
J H
JH
Deutsches Krebsforschungszentrum, Heidelberg, Federal Republic of Germany.
de Murcia
G
G
Bürkle
A
A
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000187:drug effects / Q000235:genetics; D005347:Fibroblasts / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D015723:Gene Library; D005822:Genetic Vectors; D006801:Humans; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D009243:NAD / Q000378:metabolism; D010957:Plasmids; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D016335:Zinc Fingers
D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D009243:NAD; D008769:Methylnitronitrosoguanidine; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases