Miguel Molinete

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.

Structure and function of poly(ADP-ribose) polymerase

Poly(ADP-ribose) polymerase (PARP) participates in the intricate network of systems developed by the eukaryotic cell to cope with the numerous environmental and endogenous genetoxic agents. Cloning of the PARP gene has allowed the development of genetic and molecular approaches to elucidate the structure and the function of this abundant and highly conserved enzyme. This article summarizes our present knowledge in this field.

The human poly(ADP-ribose) polymerase nuclear localization signal is a bipartite element functionally separate from DNA binding and catalytic activity.

Poly(ADP‐ribose) polymerase (PARP, EC 2.4.2.30) is a zinc finger DNA‐binding protein involved in DNA repair processes in eukaryotes. By deletion and extensive site‐directed mutagenesis, its DNA‐binding domain fused to the N‐terminus of beta‐galactosidase was shown to contain a nuclear localization signal (NLS) of the form KRK‐X(11)‐KKKSKK (residues 207–226). In vitro, both the DNA‐binding capacity and the polymerizing activity of PARP are independent of the nuclear location function. Each basic cluster is essential but not sufficient on its own for this function, while both motifs together are. Crucial basic amino acids (K207, R208 and K222) in each of these two motifs are required for nuclear homing. The results presented here support the concept that the human PARP NLS is an autonomous functional element and belongs to the class of bipartite NLSs. We show that the linear distance between the two basic clusters is not crucial. Insertional mutation analysis leading to a partial reversion of the cytoplasmic phenotype displayed by the mutant K222I highlights the crucial positioning of this lysine. The structure‐function relationship of the second cluster of basic residues is discussed.

Structure and function of the human poly(ADP-ribose) polymerase

A number of roles have been ascribed to poly(ADP-ribose) polymerase, (PARP; EC. 2.4.2.30), including involvement in DNA repair, cell proliferation, differentiation and transformation (1-4). One of our major goals is to understand the molecular basis of the complex mechanism leading to the PARP activation in response to DNA strand breaks. Cloning of the gene has allowed the development of molecular biological tools to elucidate the structure and the function(s) of this highly conserved enzyme. This paper describes the recent results obtained in our laboratory using these new approaches.