Purified poly(ADP-ribose) polymerase was inhibited by 1 ,lo-phenanthroline at pH < 8. This inhibition and the inhibition by other chelating agents suggested that this enzyme was a metalloprotein. Atomic absorption spectroscopy showed the presence of one atom of zinc per protein molecule. Dialysis of the enzyme against buffers containing 1,lO-phenanthroline resulted in the loss of activity and the removal of zinc from the enzyme. Initial rate kinetics showed that 1,lO-phenanthroline was non-competitive with NAD+ and competitive with DNA. The binding of DNA to the enzyme was unaffected by the inhibitor. These results suggest that a metal-containing site is involved as part of the interaction of DNA and poly(ADP-ribose) polymerase.Poly(ADP-ribose) polymerase covalently modifies chromatin proteins with the ADP-ribose moiety of NAD' and adds subsequent residues to form a polymer [l -31. The in uivo function of the enzyme and its product. poly(ADP-ribose), has not been defined but an involvement ,with DNA seems likely since enzymatic activity is DNA-dependent 141. The involvement of poly(ADP-ribose) polymerase in DNA repair [5,6], differentiation [7], cell growth [8], transformation [9], transcription [lo, 1 11 and the maintenance of chromatin structure 112,131 has been suggested. Despite the wealth of papers on poly(ADP-ribose) polymerase, the biological function of this enzyme is not very well understood.Poly(ADP-ribose) polymerase has been purified to near homogeneity from a variety of sources [I4 -221 and studied intensively in vitro. The overall evidence suggests that the enzymes from various sources have a similar molecular mass and that they cross-react immunologically [23,24]. It has been clearly demonstrated that the activation of polymerase requires DNA containing nicks or double-stranded breaks [25,26]. Although the DNA structures which activate the enzyme have been identified [26,27] [30,35,40]. In this communication we show that poly(AI1P-ribose) polymerase is also a zinc metalloenzyme and that zinc appears to be involved in the interaction of the enzyme and DNA. MATERIALS AND METHODS Materials Bu ffersThe extraction buffer and buffers A to C contained 50 mM Tris/HCl (pH 8.0), 1 mM EDTA, 10 mM 2-mercaptoethanol. Additional components were added to the following buffers : extraction buffer, 0.3 M NaC1, 50 mM NaHSO,, 0.5 mM dithiothreitol ; buffer A, 0.2 M NaCl, 50 mM NaHSO,, 1 mM dithiothreitol; buffer B, buffer A with 10% glycerol; buffer C, 0.25 M KCl, 10% glycerol, 5 mM dithiothreitol. Buffer D contained2 M KCl, 10% glycerol, 30 mM 2-mercaptoethanol, 5 mM dithiothreitol and 1 mM potassium phosphate (pH 7.2). Buffer E, adjusted to pH 8.0 with NH40H, contained 0.2 M (NH4)2S04,50 mM Mops and 5 mM dithiothreitol. Buffer F, used for the filter-binding assays, contained 50 mM potassium phosphate (pH 6.0), 10 mM MgCl,, 10 mM 2-mercaptoethanol. The sample buffer for sodium dodecyl sulfate (SDS)/polyacrylamide electrophoresis contained 62.5 mM Tris/HCl (pH 6.8), 2 % SDS, 10 7; glycerol, 5 % 2-mercaptoethanol a...

We have isolated an isogenic O6-methylguanine (O6-MeG)-DNA methyltransferase-defective mutant from a HeLa cell line. This mutant exhibits excess DNA strand breaks and considerable cytotoxicity after N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment. The increased frequency of strand breaks after MNNG treatment was not abolished by DNA synthesis inhibitors. We propose that the presence of unrepaired O6-MeG lesions leads to excess strand breaks and these, in turn, are mainly responsible for the cytotoxicity.

Previously it had been shown that poly(ADP‐ribose) polymerase requires DNA for its activity and that this enzyme is auto‐poly(ADP‐ribosyl)ated. The studies reported here indicate that this self‐modification inhibits the enzyme and decreases its affinity for DNA, as shown by sucrose gradient density centrifugation. The coupling of poly(ADP‐ribose) polymerase with poly(ADP‐ribose) glycohydrolase reactivates the polymerase by degrading poly(ADP‐ribose) and restoring the polymerase‐DNA complex. The assay of polymerase in the presence of glycohydrolase was made possible by use of a double‐label assay involving release of 14C‐labelled nicotinamide and the incorporation of 3H‐labelled ADP‐ribose from NAD+. These results provide the basis for a shuttle mechanism in which the polymerase can be moved on and off DNA by the action of these two enzymes. Mg2+ and histone HI appear to activate the polymerase by increasing the affinity of the polymerase for DNA.

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