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FRET Studies of DNA Polβ Mutator I260Q: The Critical Role of Fingers Domain Movements and Nucleotide Selection
Author(s) -
TowleWeicksel Jamie,
Eckenroth Brian,
Moreno Marcus,
Doublié Sylvie,
Sweasy Joann
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.879.14
Subject(s) - selection (genetic algorithm) , dna , domain (mathematical analysis) , förster resonance energy transfer , genetics , computational biology , biology , computer science , physics , quantum mechanics , fluorescence , mathematical analysis , mathematics , artificial intelligence
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 decreased levels of dNTP discrimination during binding. Our FRET results suggest that the fingers domain of I260Q fails to move during dNTP incorporation, indicating that I260Q follows a different kinetic pathway for correct dNTP incorporation compared to WT. Crystal structures also suggest that the I260Q fingers domain remains in the closed conformation and that several key residues are flexible enough within the dNTP binding pocket to allow for chemistry. Overall, this study provides new insight into the mechanism of substrate choice by a polymerase that plays a critical role in maintaining genome stability.