DNA polymerase versus DNA binding to the anticancer drug, cis-platin

Abstract

The activity of the Klenow fragment of E. coli DNA polymerase-I was inhibited in the presence of cis-diamminedichloroplatinum(II) at neutral pH in 5 mM chloride. Pre-incubations of cis-DDP with the polymerase and DNA revealed that the inhibition is primarily due to irreversible binding of the platinum complex to the enzyme. To understand the chemistry behind the inhibition, reactions of a model peptide, ERFKCPCPT and nucleotide, 5′-GMP with cis-DDP in mixtures were examined. The peptide, was selected from the DNA binding domain of human DNA polymerase-α while the mono-nucleotide serves as a model for the DNA binding. Reactions of cis-DDP with a mixture of the peptide and 5′-GMP at various concentrations ranging from equimolar to excess of nucleotide over the peptide revealed that the nucleotide can not effectively compete with the peptide. An appreciable nucleotide coordination was observed only when the nucleotide concentrations were exceeded by fourfold. At pH 6.5, the peptide complexation proceeds through the formation of an intermediate through a second order process (k=0.2 M−1 s−1) due to direct reaction between the starting dichloro-complex as well as through the aquated complex (k=>10 M−1 s−1). Platinum-195 NMR revealed that the product contains a coordination environment composed of two nitrogen and two sulfur donors consistent with the formulation that both cysteines are coordinated to and ammine ligands are retained by the metal center. Platinum(II) also readily replaced Zn(II) from the Zn-peptide complex, the latter metal ion is known to coordinate with four cysteine residues in the human DNA polymerase-α. Furthermore, the kinetics of reactions of cis-DDP and its hydrolyzed products with GpG and ApG were investigated and compared with that of peptide binding. These two dinucleotides represent the abundant binding sites in DNA reaction selected as model nucleotides. The reactions of the dichloro-platinum(II) complex with nucleotides, on the other hand, were largely controlled by the rate of aquations. The rate of first aquation process, k=1.3±0.1×10−4 s−1 evaluated from the kinetic profiles was invariant regardless of the ligands used. However, the second aquation rate constants lie in the narrow range 3–6×10−5 s−1, with GpG being favored over ApG.