Inhibition of Escherichia coli DNA polymerase I by association of the template with an N-methylisatin-β-thiosemicarbazone·copper complex

Abstract

The deoxyribonucleic acid polymerase I (deoxynucleoside triphosphate: DNA deoxynucleotidyl transferase; E.C 2.7.7.7) of Escherichia coli is inhibited more than 99% by 50 μM N-methylisatin-β-thiosemicarbazone (M-IBT) + 50 μM CuSO₄. When added alone, 50 μM M-IBT enhanced activity, whereas 50 μM CuSO₄ alone inhibits the reaction by 40%. Inhibition is reversed by the competing chelating agent ethylenediamine tetraacetate. A related compound, 1-formylisoquinoline thiosemicarbazone, also inhibits enzyme activity in the presence of cupric ions.

Polymerase inhibition by M-IBT + CuSO₄ is relieved when increasing amounts of nucleic acid, but not increasing amounts of enzyme, are added. This suggests that the DNA template, not the polymerase, is the target of inhibition by M-IBT + CuSO₄.

The biological pertinence of nucleic acid as a target for the M-IBT + CuSO₄ complex is shown by the binding of Rous sarcoma virus (RSV) 70 S RNA to the complex formed within the intact virus particle. The importance of metal ions for this drug action is supported by the ability of ethylenediamine tetraacetate to reverse the binding.