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

Journal of Biological Chemistry (Impact Factor: 4.57). 04/2014; 289(23). DOI: 10.1074/jbc.M114.561878
Source: PubMed


During DNA repair, DNA polymerase beta (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). In order 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 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.

Download full-text


Available from: Christal D Sohl, Oct 28, 2015
  • [Show abstract] [Hide abstract]
    ABSTRACT: DNA-dependent DNA polymerases are the main enzymes that catalyze DNA replication. Higher eukaryotic cells have 19 DNA polymerases with strikingly different properties [1]. Mitochondrial DNA polymerase γ of the A family and most of the nuclear enzymes of the B family are high-fidelity DNA polymerases that are involved not only in genomic DNA replication but also in DNA repair. Among the other 15 proteins, DNA polymerases belonging to the X and Y families have a special place. The majority of these enzymes are also involved in repair, including base excision repair and nonhomologous end joining. Some of them play a specific role in replication of damaged DNA templates. This process is referred to as translesion synthesis (TLS). DNA polymerases β and λ, which belong to the X structural family, are polyfunctional enzymes; their properties and functions are discussed.
    No preview · Article · Dec 2010 · Molecular Biology
  • [Show abstract] [Hide abstract]
    ABSTRACT: DNA polymerases and substrates undergo conformational changes upon forming protein-ligand complexes. These conformational adjustments can hasten or deter DNA synthesis and influence substrate discrimination. From structural comparison of binary DNA and ternary DNA/dNTP complexes of DNA polymerase β, several side-chains have been implicated in facilitating formation of an active ternary complex poised for chemistry. Site-directed mutagenesis of these highly conserved residues (Asp192, Arg258, Phe272, Glu295, and Tyr296) and kinetic characterization provides insight into the role these residues play during correct and incorrect insertion as well as their role in conformational activation. The catalytic efficiencies for correct nucleotide insertion for alanine mutants was wild type ≈ R258A > F272A ≈ Y296A > E295A > D192. Since the efficiencies for incorrect insertion was affected to about the same extent for each mutant, effects on fidelity were modest (<5-fold). The R258A mutant exhibited an increase in the single-turnover rate of correct nucleotide insertion. This suggests that the wild-type Arg258 side-chain generates a population of non-productive ternary complexes. Structures of binary and ternary substrate complexes of the R258A mutant and a mutant associated with gastric carcinomas, E295K, provide molecular insight into intermediate structural conformations not appreciated previously. While the R258A mutant crystal structures were similar to wild-type enzyme, the open ternary complex structure of E295K indicates that Arg258 stabilizes a non-productive conformation of the primer terminus that would decrease catalysis. Significantly, the open E295K ternary complex binds two metal ions indicating that metal binding cannot overcome the modified interactions that have interrupted the closure of the N-subdomain.
    No preview · Article · Sep 2014 · Journal of Biological Chemistry
  • [Show abstract] [Hide abstract]
    ABSTRACT: Among the set of mammalian DNA polymerases, DNA polymerases belonging to the X and Y families have a special place. The majority of these enzymes are involved in repair, including base excision repair and non-homologous end joining. Some of them play a crucial role during the specific process which is referred to as translesion synthesis (TLS). TLS intends for the cell surviving during the replication of damaged DNA templates. Additionally, specific activities of TLS-polymerases have to be useful for repair of double-stranded clustered lesions: if the synthesis is proceeded via base excision repair process, the role of DNA polymerases β or λ will be important. In this review we discussed the biochemical properties and functional relevance of X family DNA polymerases β and λ. Copyright © 2015 Elsevier B.V. All rights reserved.
    No preview · Article · Feb 2015 · DNA Repair
Show more