DNTP-Dependent Conformational Transitions in the Fingers Subdomain of Klentaq1

Abstract

DNA polymerases are responsible for the accurate replication of DNA. Kinetic studies indicate the requirement for multiple intermediate enzyme conformations in this process. Structural studies show a large conformational change in the fingers subdomain of DNA polymerase on binding of a correct dNTP. Using single molecule FRET we show that the conformational transition affecting the fingers subdomain also takes place in the apo and DNA-bound forms of the enzyme. In addition a third conformation is observed which is occupied in the presence of dNTP alone, and in the presence of a non base pairing dNTPs. The relative proportions of the identified states are altered dramatically depending on substrate. Binding of the correct nucleotide displaces this equilibrium dramatically towards the closed form, while binding of an incorrect nucleotide favors a more open conformation. The results suggest that the closed state is by design less energetically favored, providing a thermodynamic brake on incorrect nucleotide insertion.