Overproduction of the poly(ADP-ribose) polymerase DNA-binding domain blocks alkylation-induced DNA repair synthesis in mammalian cells.

Abstract

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.