Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Record number 509793
Title Probing the Conformational Landscape of DNA Polymerases Using Diffusion-Based Single-Molecule FRET
Author(s) Hohlbein, J.; Kapanidis, A.N.
Source In: Methods in Enzymology / Spies, Maria, Chemla, Yann R., Apple Academic Press Inc (Methods in Enzymology ) - ISBN 9780128092675 - p. 353 - 378.
DOI https://doi.org/10.1016/bs.mie.2016.08.023
Department(s) Biophysics
VLAG
EPS
Publication type Peer reviewed book chapter
Publication year 2016
Keyword(s) Confocal microscopy - DNA polymerase - Single-molecule Förster resonance energy transfer
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.

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