Details

Autor(en) / Beteiligte

• Schärfen, Leonard
• Schlierf, Michael

Titel

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

Ist Teil von

• Methods (San Diego, Calif.), 2019-10, Vol.169, p.11-20

Ort / Verlag

United States: Elsevier Inc

Erscheinungsjahr

2019

Link zum Volltext

Quelle

Alma/SFX Local Collection

Beschreibungen/Notizen

• •DNA forms sequence-dependent secondary structures.•Proteins compete for DNA binding and often induce conformational changes.•Single-molecule FRET allows real-time monitoring of conformations and conformational dynamics.•Unbiased analysis using hidden Markov models or student’s t-test based models reveals conformational states.•Single-molecule experiments allow bottom up reconstructions of protein competitions to reveal higher order regulations. 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.