Overproduction of the poly(ADP-ribose) polymerase DNA-binding domain blocks alkylation-induced DNA repair synthesis in mammalian cells.
The zinc-finger DNA-binding domain (DBD) of poly (ADP-ribose) polymerase (PARP, EC 2.4.2.30) specifically recognizes DNA strand breaks induced by various DNA-damaging agents in eukaryotes. This, in turn, triggers the synthesis of polymers of ADP-ribose linked to nuclear proteins during DNA repair. The 46 kDa DBD of human PARP, and several derivatives thereof mutated in its first or second zinc-finger, were overproduced in Escherichia coli, in CV-1 monkey cells or in human fibroblasts to study their DNA-binding properties, the trans-dominant inhibition of resident PARP activity, and the consequences on DNA repair, respectively. A positive correlation was found between the in vitro DNA-binding capacity of the recombinant DBD polypeptides and their inhibitory effect on PARP activity stimulated by the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Furthermore, overproduced wild-type DBD blocked unscheduled DNA synthesis induced in living cells by MNNG treatment, but not that induced by UV irradiation. These results define a critical role for the second zinc-finger of PARP for DNA single-stranded break binding and furthermore underscore the importance for PARP to act as a critical regulatory component in the repair of DNA damage induced by alkylating agents.
Institut de Biologie Moléculaire et Cellulaire du Centre National de la Recherche Scientifique associé à l'Université Louis Pasteur, Strasbourg, France.
M
M
Molinete
W
W
Vermeulen
A
A
Bürkle
J
J
Ménissier-de Murcia
J H
JH
Küpper
J H
JH
Hoeijmakers
G
G
de Murcia
D000478:Alkylation; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis* / Q000187:drug effects / Q000378:metabolism; D004260:DNA Repair; D004926:Escherichia coli; D000882:Haplorhini; D006801:Humans; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D016335:Zinc Fingers
D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011994:Recombinant Proteins; D008769:Methylnitronitrosoguanidine; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases