Correlation of the Kinetics of Finger Domain Mutants in RB69 DNA Polymerase with Its Structure

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Abstract

We have estimated pre-steady-state kinetic parameters for the addition of a single nucleotide residue by a set of RB69 DNA polymerase mutants in which four highly conserved residues in the fingers domain have been replaced by Ala. The relationship between the kinetic constants exhibited by the mutants and the structure of the ternary complex [Franklin, M., Wang, J., and Steitz T. (2001) Cell 105, 657−667] was consistent with the following sets of interactions between the conserved residues and oxygen atoms in the triphosphate portion of the incoming dNTP: (i) the ε-amino group of K560 contacts oxygen atoms of the α- and γ-phosphates, (ii) the amide side chain of Asn 564 forms a hydrogen bond via a water molecule with the nonbridging oxygen of the β-phosphate, and (iii) the ε-amino and δ-guanidino groups of K486 and R482, respectively, contact the nonbridging oxygens of the γ-phosphate. We have also determined the pre-steady-state kinetic parameters for the addition of both dCTP and dCDP onto a 13/20mer primer/template with an exo- derivative of RB69 DNA polymerase and have shown that the deoxynucleoside diphosphate can be incorporated, in contrast to the behavior of the Klenow fragment which cannot use dCDP as a substrate. We have shown that, with RB69 DNA polymerase, in contrast to the Klenow fragment, there is no inhibition of the primer-extension reaction by incoming NTPs having either noncomplementary bases or ribo- instead of a deoxyribose moieties. This implies that the mode of recognition of incoming dNTPs and triggering of the conformational change, which is thought to occur prior to the chemical step, differs between these two enzymes.

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