Abstract

Abstract The Escherichia coli DNA polymerase, in the absence of DNA, contains a single binding site for which all the deoxyribonucleoside 5'-triphosphates complete. Dissociation constants of the enzyme-triphosphate complexes range from 1 x 10-5 to 2 x 10-4 m. Various analogues of the deoxyribonucleoside 5'-triphosphates also bind to the same site on DNA polymerase. The principal structural requirement for detectable binding at this site appears to be possession of a triphosphate group. Binding of triphosphates is inhibited when the concentration of potassium phosphate is increased, and is inhibited, but to a lesser extent, by increasing potassium chloride concentration. In addition, binding is abolished in the absence of Mg++ and in the presence of 9 mm EDTA. The strength of binding decreases with increasing temperature. Binding measurements were made with equilibrium dialysis microcells, which require only 20 µl of protein solution and in which equilibrium with triphosphates is attained within 90 min at 22°.