Probing the Conformational Landscape of DNA Polymerases Using Diffusion-Based Single-Molecule FRET

J. Hohlbein*, A.N. Kapanidis

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapterAcademicpeer-review

5 Citations (Scopus)

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.

Original languageEnglish
Title of host publicationMethods in Enzymology
Subtitle of host publicationSingle-Molecule Enzymology: Fluorescence-Based and High-Throughput Methods
EditorsMaria Spies, Yann R. Chemla
PublisherApple Academic Press Inc
Pages353-378
Volume581
ISBN (Print)9780128092675
DOIs
Publication statusPublished - 2016

Publication series

NameMethods in Enzymology
Volume581
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • Confocal microscopy
  • DNA polymerase
  • Single-molecule Förster resonance energy transfer

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