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.

Overexpression of dominant negative PARP interferes with tumor formation of HeLa cells in nude mice: Evidence for increased tumor cell apoptosis in vivo

“…In these so-called COM3 cells, we could show that expression of dominant negative PARP sensitizes cells against g-irradiation and the alkylating agent MNNG, while there was neither an in¯uence detectable on DNA replication nor on cellular proliferation in the absence of genotoxic stress (KuÈ pper et al, 1995). This sensitization is associated with a block in DNA base excision repair (Molinete et al, 1993) and increased gene ampli®cation and sister chromatid exchanges (KuÈ pper et al, 1995Schreiber et al, 1995). We were then interested to see whether PARP DBD overexpression a ects tumor formation in nude mice.…”

Potentiation of anti-cancer agent cytotoxicity by the potent poly(ADP-ribose) polymerase inhibitors NU1025 and NU1064

“…through overexpression of the DBD. also sensitizes cells to y-irradiation and alkylating acgents (Molinete et al 1993: Kupper et al 1995. an observation that is consistent with the effects of the benzamides being primarily caused by PARP inhibition.…”

DNA damage and repair in tumour and non-tumour tissues of mice induced by nicotinamide

“…Horsman et al (1993 showed that there is no significant difference between the radiosensitising effect of NAM at doses of 100 mg kg-' and 1000 mg kg-1 in a C3H mammary carcinoma, even though the difference in plasma and tumour concentrations of NAM was 5-to 10-fold. These data have suggested an alternative explanation for the radiosensitising properties of NAM; namely that NAM at tumour concentrations of 100 juM or higher inhibits the PARP enzyme (IC50= 30 -100 gIM) (Molinete et al, 1993), which has been shown to be involved in DNA repair (Satho and Lindahl, 1994). The induced DNA damage at NAM doses of 100 and 1000 mg kg-' could be a result of PARP inhibition because of high tumour tissue levels of NAM (Figure 2), which in turn resulted in elevated NAD levels ( Figure 3) and accumulation of DNA damage (Figure 1).…”

“…The PARP enzyme is a DNAbinding enzyme with two zinc fingers in the DNA-binding domain (De Murcia et al, 1989;Molinete et al, 1993). The enzyme activity has been associated with DNA repair, apoptosis, cell proliferation, cell differentiation and genome surveillance (Althaus and Richter, 1987;Satoh et al, 1994;Kaufman et al, 1993;Dai et al, 1987).…”