Interaction of DNA polymerase I of Escherichia coli with nucleotides. Antagonistic effects of single-stranded polynucleotide homopolymers

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Abstract
Binding of deoxyribonucleoside 5''-triphosphates to DNA polymerase I of E. coli was measured by using a microscale nonequilibrium dialysis method. It allowed rapid and economic measurement of dissociation constants, with negligible interfering side reactions. A stoichiometry of 1 mol of nucleoside 5''-triphosphate/mol of DNa polymerase was measured, and the occurrence of a single binding site was established, for which the nucleotides competed in the binary complex with the polymerase. Binding affinities decreased in the order dGTP .gtorsim. dATP > dCTP .simeq. dTTP. These results are in agreement with previous finding [P. T. Englund, J. A. Huberman, T. M. Jovin and A. Kornberg (1969)] except that, in a few cases, values of dissociation constants were smaller by factors of 2-3. The cations Mg2+ and Mn2+, as well as spermine, slightly enhanced complex stability at low levels and decreased it at high concentrations, while NaCl and Hg2+ had only destabilizing effects. Recognition between nucleoside 5''-triphosphates and nuelotide templates was studied by titration of the polymerase-[3H]dGTP complex with polynucleotide homopolymers. Complementary poly(dC) did not affect binding of dGTP, and noncomplementatry templates caused rejection of the nucleotide. Rejection of dGTP followed a saturation dependence with an equivalence of 110 .+-. 10 monomer units of polynucleotides bound per molecule of DNA plymerase. The results favor a model by which recognition arises chiefly from the stereogeometrical fit of complementary template and nucleoside 5''-triphosphate into a riged binding site.

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