Kinetic measurement of 2-aminopurine X cytosine and 2-aminopurine X thymine base pairs as a test of DNA polymerase fidelity mechanisms.

Abstract

Enzyme kinetic measurements are presented showing that Km rather than Vmax discrimination governs the frequency of forming 2-aminopurine-cytosine base mispairs by [human epidermoid carcinoma KB cell] DNA polymerase .alpha.. An in vitro system is used in which incorporation of dTMP or dCMP occurs opposite a template 2-aminopurine, and values for Km and Vmax are obtained. Results from a previous study in which dTTP and dCTP were competing simultaneously for insertion opposite 2-aminopurine indicated that dTMP is inserted 22 times more frequently than dCMP. The ratio of Km values .**GRAPHIC**. = 25 .+-. 6, which agrees quantitatively with the dTMP/dCMP incorporation ratio obtained previously. .**GRAPHIC**. is indistinguishable from .**GRAPHIC**. These Km and Vmax data are consistent with predictions from a model, the Km discrimination model, in which replication fidelity is determined by free energy differences between matched and mismatched base pairs. Central to this model is the prediction that the ratio of Km values for insertion of correct and incorrect nucleotides specifies the insertion fidelity, and the maximum velocities of insertion are the same for both nucleotides.