Real-time monitoring of protein-induced DNA conformational changes using single-molecule FRET

Research output: Contribution to journalResearch articleContributedpeer-review

Contributors

Abstract

Apart from being storage devices for genetic information, nucleic acids can provide regulatory structures through evolutionarily optimized sequences. The interaction of proteins binding specifically to such sequences and resulting secondary structures, or the exposure of single-stranded DNA add a versatile regulatory framework for cells. Biochemical and structural biology experiments have revealed important underlying concepts of protein-DNA interactions but are often limited by ensemble averaging or static information. To decipher the dynamics of conformations adopted by protein-DNA complexes, single-molecule approaches have become a powerful resource over the past two decades. In particular single-molecule FRET (smFRET), which allows a read-out of DNA or protein conformations, became widely used. Here, we illustrate how to implement the technique and exemplarily describe how smFRET yields insights into conformational changes of DNA secondary structures induced by the single-stranded DNA binding protein SSB. We further explain how we use smFRET to study mechanisms of the replication initiator DnaA and the competition of DnaA and SSB for single-stranded DNA. We anticipate that smFRET will further develop into a particularly useful technique to study dynamic competitions of proteins for the same DNA substrate.

Details

Original languageEnglish
Pages (from-to)11-20
Number of pages10
JournalMethods
Volume169
Publication statusPublished - 1 Oct 2019
Peer-reviewedYes

External IDs

Scopus 85062086264
ORCID /0000-0002-6209-2364/work/142237623

Keywords

Keywords

  • Single-molecule FRET, Protein-DNA interactions, Competition, Conformational dynamics, State identification, Hidden Markov models, INDUCED FLUORESCENCE ENHANCEMENT, RESONANCE ENERGY-TRANSFER, STRANDED-DNA, ESCHERICHIA-COLI, MAGNETIC TWEEZERS, BINDING-PROTEIN, REPLICATION, INITIATION, DYNAMICS, MECHANISM