Fluorescence Resonance Energy Transfer Studies of DNA Polymerase β

• Published in: The Journal of biological chemistry Vol. 289; no. 23; pp. 16541 - 16550
• Main Authors: Towle-Weicksel, Jamie B., Dalal, Shibani, Sohl, Christal D., Doublié, Sylvie, Anderson, Karen S., Sweasy, Joann B.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 06.06.2014
• Subjects: Base Excision Repair (BER); DNA Polymerase; Fidelity of DNA Synthesis; Fluorescence Resonance Energy Transfer (FRET); Mutagenesis; Protein Conformation; Protein Conformation; DNA Polymerase; Fluorescence Resonance Energy Transfer (FRET); Mutagenesis; Fidelity of DNA Synthesis; Base Excision Repair (BER)
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.561878

During DNA repair, DNA polymerase β (Pol β) is a highly dynamic enzyme that is able to select the correct nucleotide opposite a templating base from a pool of four different deoxynucleoside triphosphates (dNTPs). To gain insight into nucleotide selection, we use a fluorescence resonance energy transfer (FRET)-based system to monitor movement of the Pol β fingers domain during catalysis in the presence of either correct or incorrect dNTPs. By labeling the fingers domain with (((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS) and the DNA substrate with Dabcyl, we are able to observe rapid fingers closing in the presence of correct dNTPs as the IAEDANS comes into contact with a Dabcyl-labeled, one-base gapped DNA. Our findings show that not only do the fingers close after binding to the correct dNTP, but that there is a second conformational change associated with a non-covalent step not previously reported for Pol β. Further analyses suggest that this conformational change corresponds to the binding of the catalytic metal into the polymerase active site. FRET studies with incorrect dNTP result in no changes in fluorescence, indicating that the fingers do not close in the presence of incorrect dNTP. Together, our results show that nucleotide selection initially occurs in an open fingers conformation and that the catalytic pathways of correct and incorrect dNTPs differ from each other. Overall, this study provides new insight into the mechanism of substrate choice by a polymerase that plays a critical role in maintaining genome stability. DNA Pol β participates in base excision repair by choosing correct dNTP to fill single-nucleotide gaps in DNA. Pol β experiences a non-covalent step with correct dNTP selection. Correct and incorrect dNTP incorporation by Pol β are different. FRET-based system of Pol β elucidates a mechanism of substrate choice necessary for understanding the molecular basis of human disease.