A Comparative Study of the Effect of Normal Substrates and 5-Iodo-2′-deoxyuridine Triphosphate, a Metabolic Analog of Thymidine Triphosphate, on the Inactivation of Escherichia coli Deoxyribonucleic Acid Polymerase I and II by Ultraviolet Irradiation

The ultraviolet inactivation of DNA polymerase I and II both followed first order kinetics. There was no alteration in enzyme properties such as optimum concentration of magnesium chloride or of DNA template-primer, pH profile, or substrate saturation curve, and hence is compatible with an all-or-none effect. The rate of ultraviolet inactivation of DNA polymerase II was about 3 times that of DNA polymerase I, and this difference was further augmented when 5-iodo-2'-deoxyuridine 5'-triphosphate (IdUTP), a metabolic analog of deoxythymidine triphosphate, was included during irradiation. The enhanced ultraviolet inactivation observed in the presence of IdUTP of both DNA polymerase I and II was not only dependent on the IdUTP concentration but also was a saturable phenomenon. 5-Iodo-2'-deoxyuridine and sodium iodide sensitized these enzymes to ultraviolet radiations; however, the enhanced rate of inactivation by ultraviolet light was linear over the concentrations used. Magnesium chloride by itself had no effect on the inactivation of either DNA polymerase I or II; however, magnesium chloride further enhanced the sensitizing effect of IdUTP and of 5-iodo-2'-deoxyuridine but not of sodium iodide. The optimum magnesium chloride concentration for such an effect was identical with that required for optimum catalytic activity. The molar ratio of magnesium chloride to IdUTP was not crucial for this effect. The DNA template also sensitized the polymerases toward inactivation by ultraviolet irradiation, the effect being greater with DNA polymerase II relative to DNA polymerase I. DNA further increased the IdUTP-sensitizing effect to ultraviolet radiation to the same extent as magnesium chloride. The free radical intermediate postulation proposed previously (Cysyk, R., and Prusoff, W. H. (1972) J. Biol. Chem. 247, 2522) for the 5-iodo-2'-deoxyuridine ultraviolet inactivation of thymidine kinase was also considered for the present IdUTP-DNA polymerase system.