Gérard Gradwohl

Zinc-binding domain of poly(ADP-ribose)polymerase participates in the recognition of single strand breaks on DNA.

Poly(ADP-ribose)polymerase is a chromatin-associated enzyme of eukaryotic cell nuclei that catalyses the covalent attachment of ADP-ribose units from NAD+ to various nuclear acceptor proteins. This post-translational modification has been postulated to influence several chromatin functions, particularly those where nicking and rejoining of DNA occur. Poly(ADP-ribosyl)ation reactions are strictly dependent upon the presence of interruptions on DNA. We have recently demonstrated that the DNA-binding domain of the protein containing two putative zinc-fingers binds DNA in a zinc-dependent manner. The basis for the recognition of the DNA strand breaks by this enzyme, and more precisely, its 29,000 Mr N-terminal part, which contains the metal binding sites, needed to be clarified. DNA probes harbouring a single strand interruption at a defined position were constructed from synthetic oligonucleotides. DNase I protection studies show that poly(ADP-ribose)polymerase specifically binds to a DNA single-strand break by its metal-binding domain depending upon the presence of Zn(II). These results support the idea that the enzyme participates to the maintenance of DNA integrity in eukaryotes.

Zinc-binding proteins detected by protein blotting

The Western blotting technique was used for the detection of zinc-binding proteins. Proteins were separated electrophoretically on 15% polyacrylamide-sodium dodecyl sulfate minigels, the gels were soaked in a reduction buffer, and the proteins were transferred to nitrocellulose filters. Zinc-binding proteins were probed with radioactive zinc (65Zn) and were detected by autoradiography. This technique allows the detection of as little as 20 to 100 pmol of zinc metalloproteins.

Poly(ADP-ribose) polymerase forms loops with DNA

The interaction between highly purified poly(ADP-ribose) polymerase from calf thymus and different topological forms of pBR322 DNA has been studied by gel retardation electrophoresis and electron microscopy. We show that: (i) in the absence of nicks on DNA the enzyme has a marked affinity for supercoiled (form I) DNA, (ii) in the presence of single stranded breaks poly(ADP-ribose) polymerase preferentially binds to form II, (iii) in all cases enzyme molecules are frequently located at DNA intersections, (iv) a cooperative binding of the enzyme on DNA occurs.

Expression in E.coli of the catalytic domain of rat poly(ADP‐ribose)polymerase

A 2 kilobase pair cDNA coding for the entire C‐terminal catalytic domain of rat poly(ADP‐ribose)polymerase has been expressed in E. coli. The overproduced 55 kDa polypeptide is active in synthesizing poly(ADP‐ribose) and the 4 kDa N‐terminal region of this domain is recognized by the monoclonal antibody C I,2 directed against the calf enzyme. Also, the minor αchymotrypsin cleavage site found in the human catalytic domain is not present in the rat enzyme as revealed by the absence of the 40 kDa specific degradation product in the E. coli cells expressing the rat domain. The expression of this partial rat cDNA should thus permit the rapid purification and subsequent crystallization of the catalytic domain of the enzyme.

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.

Poly(ADP—Ribose)Polymerase in Xenopus laevis

Among the different biological systems in which poly(ADP-ribosyl)ation has been studied, cell lines have been widely used, especialy to investigate the role of poly(ADP-ribose)polymerase (PARP) in DNA repair(1). Other systems such as regenerating rat liver (2) or mitogen stimulated lymphocytes (3) permitted several authors to suggest the implication of the PARP in DNA replication and cell proliferation. Even so, one has to admit that the precise biological function of poly(ADP-ribosyl)ation of proteins acceptors is still unclear.

Localization of the Zinc-Binding Sites in the DNA-Binding Domain of the Bovine Poly(ADP-Ribose) Polymerase

The poly(ADP-ribose) polymerase is a chromatin associated enzyme of eukaryotic cell nuclei, which has an absolute requirement for DNA (1). In vivo (2, 3) and in vitro (4, 5) its activity is stimulated by DNA containing nicks or double stranded breaks. The binding of the enzyme to sites of strand breaks on DNA, is a prerequisite step to activation. Like other classes of proteins involved in nucleic acid binding poly(ADP-ribose) polymerase is a zinc metalloenzyme (6) and it is suggested that a metal-binding site is involved as part of the interaction of DNA and the enzyme. Kameshita et al. (7) elucidated the localization of three functional domains in the enzyme molecule. These domains are separable by mild proteolysis, the NH2 fragment of 46 kDa corresponds to the DNA-binding domain, the COOH- terminal fragment of 54 K is the domain for the substrate NAD+ binding, and the third one of 22 K contains the sites for accepting poly(ADP-ribose). We attempted to localize the zinc binding sites, having regard to the DNA- binding domain of the enzyme. Radioactive zinc (65Zn) and 32P-labelled nick translated DNA were used alternatively, to analyze electro-blots loaded with proteolytic fragments of the enzyme. The same blots were further immunostained with monoclonal antibodies. Our results showed that the radioactive zinc is only bound to the proteolytic fragments containing the DNA-binding domain of the enzyme.

Poly (ADP-Ribosyl) Ation Reactions and Modulation of Chromatin Structure

Posttranslational modifications of histones are generally considered as potential modulators of chromatin structure during DNA transcription and replication. One of them, the addition of poly(ADP-ribose) is ubiquitous to eukaryotes and is mediated at the expense of the NAD pool by the chromatin bound enzyme, poly ADP-ribose polymerase. This highly conserved enzyme is strictly DNA-dependent and is inactive unless stimulated by DNA strand breaks (Ueda and Hayaishi, 1985; Gaal and Pearson, 1985; Althaus and Richter, 1987).

Poly(ADP-Ribose) Polymerase: DNA Complexes Visualized as Looped Structures by Electron Microscopy

Poly(ADP-ribose) polymerase is a chromatin bound enzyme which catalyzes the covalent attachment of ADP-ribose units from the coenzyme NAD to various nuclear proteins (1–3). Poly(ADP-ribosyl)ation is a posttranslational modification which appears to be involved in DNA excision repair, cellular proliferation and differentiation (1–3) and in modulation of chromatin structure (4, 5). This DNA dependent enzyme is inactive unless stimulated by DNA strand breaks. Although the DNA structures which activate the enzyme have been identified (6, 7) the basis for the DNA requirement as well as the stimulation of the enzyme activity are not yet understood. We have studied the interaction between poly(ADP- ribose) polymerase and different DNAs using electron microscopy and gel retardation electrophoresis.