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 126.96.36.199) 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.
|Keywords||*DNA Repair, 0 (Recombinant Proteins), 70-25-7 (Methylnitronitrosoguanidine), 9007-49-2 (dna), Alkylation, Amino Acid Sequence, Animals, Binding Sites, Cell Line, Cells, Cultured, DNA/*biosynthesis/drug effects/metabolism, EC 188.8.131.52 (Poly(ADP-ribose) Polymerases), Escherichia coli, Haplorhini, Human, Methylnitronitrosoguanidine/pharmacology, Molecular Sequence Data, Mutation, Poly(ADP-ribose) Polymerases/antagonists & inhibitors/genetics/*metabolism, Recombinant Proteins/metabolism, Support, Non-U.S. Gov't, Transfection, Zinc Fingers|
Molinete, M., Vermeulen, W., Bürkle, A., Ménissier-de Murcia, J., Küpper, J.H., Hoeijmakers, J.H.J., & de Murcia, G.. (1993). Overproduction of the poly(ADP-ribose)polymerase DNA-binding domain blocks alkylation-induced DNA repair synthesis in mammalian cells.. EMBO Journal, 12, 2109–2117. Retrieved from http://hdl.handle.net/1765/3050