title: Probing the Conformational Landscape of DNA Polymerases Using Diffusion-Based Single-Molecule FRET.
abstract: Monitoring conformational changes in DNA polymerases using single-molecule Förster resonance energy transfer (smFRET) has provided new tools for studying fidelity-related mechanisms that promote the rejection of incorrect nucleotides before DNA synthesis. In addition to the previously known open and closed conformations of DNA polymerases, our smFRET assays utilizing doubly labeled variants of Escherichia coli DNA polymerase I were pivotal in identifying and characterizing a partially closed conformation as a primary checkpoint for nucleotide selection. Here, we provide a comprehensive overview of the methods we used for the conformational analysis of wild-type DNA polymerase and some of its low-fidelity derivatives; these methods include strategies for protein labeling and our procedures for solution-based single-molecule fluorescence data acquisition and data analysis. We also discuss alternative single-molecule fluorescence strategies for analyzing the conformations of DNA polymerases in vitro and in vivo.
authors: Laboratory of Biophysics, Wageningen University and Research, Wageningen, The Netherlands; Microspectroscopy Centre, Wageningen University and Research, Wageningen, The Netherlands. Electronic address: johannes.hohlbein@wur.nl.
authors: J
authors: J
authors: Hohlbein
authors: Clarendon Laboratory, University of Oxford, Oxford, United Kingdom. Electronic address: kapanidis@physics.ox.ac.uk.
authors: A N
authors: AN
authors: Kapanidis
mesh_terms: D004247:DNA / Q000737:chemistry*; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000302:isolation & purification*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D036103:Nanotechnology; D011487:Protein Conformation; D000072760:Single Molecule Imaging / Q000379:methods*
chemical_list: D004247:DNA; D004259:DNA-Directed DNA Polymerase
keywords: Confocal microscopy; DNA polymerase; Single-molecule Förster resonance energy transfer