Destabilization of Zn2+ coordination in ADP‐ribose transferase (polymerizing) by 6‐nitroso‐1,2‐benzopyrone coincidental with inactivation of the polymerase but not the DNA binding function

Abstract

6-Nitroso- 1,2-benzopyrone, an oxidation product of 6-amino- 1,2-benzopyrone, binds to the DNA-recognizing domain of the ADP-ribose transferase protein and preferentially destabilizes Zn²⁺ from one of the two zinc finger polypeptide complexes present in the intact enzyme, as determined by the loss of 50% of ⁶⁵Zn²⁺ from the ⁶⁵Zn²⁺-isolated protein molecule, coincidental with the loss of 99% of enzymatic activity. The 50% zinc-deficient enzyme still binds to a DNA template. consisting of a 17-mer DNA primer annealed to M 13 positive strand, resulting in the blocking of DNA synthesis by the Klenow fragment of Pol I, Auto-poly-ADP-ribosylated ADP-ribose transferase, which is the probable physiological state of this protein in intact cells, does not bind to primer-template DNA and does not block DNA synthesis by the Klenow fragment. On the basis of this in vitro model it is proposed that molecules which inhibit or inactivate ADP-ribose transferase in intact cells can induce significant alteration in DNA structure and replication.