FRET Studies of DNA Polβ Mutator I260Q: The Critical Role of Fingers Domain Movements and Nucleotide Selection

A highly dynamic enzyme, Polβ selects the correct nucleotide opposite a templating base from a pool of four different deoxynucleoside triphosphates (dNTPs) during DNA repair. Our objective is to study the mechanism of dNTP selection. We used fluorescence resonance energy transfer (FRET) to monitor movement of the Polβ fingers domain during base-catalysis in the presence of correct or incorrect dNTP. We provided evidence of the presence of a non-covalent step that takes place after closing of the fingers domain, but before chemistry, and that the fingers domain does not close in the presence of incorrect dNTP. This suggests that Polβ monitors fidelity before the fingers close, and that incorporation of incorrect dNTP is likely to follow a different kinetic pathway from correct. Currently, we are studing the movements of the mutator I260Q to further demonstrate that dNTP selection occurs at ground state binding. I260Q is located in the hinge region of Polβ and has similar kinetics to WT, but experiences dec...