Inhibition of Escherichia coli DNA polymerase-I by the anti-cancer drug cis-diaminedichloroplatinum(II): what roles do polymerases play in cis-platin-induced cytotoxicity?

Activities of Escherichia coli DNA polymerase-I were examined in the presen ce of the anti-tumor drug cis-diaminedichloroplatinum(II) and its inactive geometric isomer tr trans-diaminedichloroplatinum(II), The trans-isomer did not inhibit the enzyme activity. The anti-tumor drug, on the other hand, r etarded the enzyme in its ability to extend the primer strand of DNA, Two a lternative mechanisms of inhibition, covalent binding of cis-diaminedichlor oplatinum(II) to the polymerase and to the template DNA, were explored. Sel ective preincubations of the platinum drug with the polymerase and DNA reve al that the inhibition is primarily due to covalent binding to the enzyme. The rates of inhibition were found to be first order in enzyme and zeroth o rder in platinum in the concentration range 0.05-3.0 mM, A mechanism that d eals with the formation of an initial platinum-polymerase-I complex with a binding constant > 10(5) M-1 followed by a further reaction to form an inhi bitory complex is consistent with the kinetic data, The rate limiting first order rate constant for the formation of the inhibitory complex is compara ble to that observed for the thiol coordination of peptides containing cyst eine residues. Analyses of known structures and functions of catalytic doma ins of various polymerases point to the direction that the inhibition is pe rhaps due to the distortion of the DNA binding domain of the enzyme due to platinum coordination. (C) 1999 Federation of European Biochemical Societie s.