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

Abstract

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.