Fluorescence resonance energy transfer studies of DNA polymerase β: the critical role of fingers domain movements and a novel non-covalent step during nucleotide selection.
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.
From the Departments of Therapeutic Radiology and.
Jamie B
JB
Towle-Weicksel
From the Departments of Therapeutic Radiology and.
Shibani
S
Dalal
Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520 and.
Christal D
CD
Sohl
the Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont 05405.
Sylvie
S
Doublié
Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520 and.
Karen S
KS
Anderson
From the Departments of Therapeutic Radiology and joann.sweasy@yale.edu.
Joann B
JB
Sweasy
D001483:Base Sequence; D019951:DNA Polymerase beta / Q000737:chemistry*; D017931:DNA Primers; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D009711:Nucleotides / Q000737:chemistry*
D017931:DNA Primers; D009711:Nucleotides; D019951:DNA Polymerase beta
Base Excision Repair (BER); DNA Polymerase; Fidelity of DNA Synthesis; Fluorescence Resonance Energy Transfer (FRET); Mutagenesis; Protein Conformation