Single-Molecule Studies of ssDNA-Binding Proteins Exchange

Methods Enzymol. 2018;600:463-477. doi: 10.1016/bs.mie.2017.11.017. Epub 2018 Feb 1.

Abstract

Single-stranded DNA-binding protein (SSB) is important not only for the protection of single-stranded DNA (ssDNA) but also for the recruitment of other proteins for DNA replication, recombination, and repair. The interaction of SSB with ssDNA is highly dynamic as it exists as an intermediate during cellular processes that unwind dsDNA. It has been proposed that SSB redistributes itself among multiple ssDNA segments, but transient intermediates are difficult to observe in bulk experiments. We can use single-molecule FRET microscopy to observe intermediates of the transfer of a single Escherichia coli SSB from one ssDNA strand to another or exchange of one SSB for another on a single ssDNA in real time. This single-molecule approach can be further applicable to understand relative binding affinities and competitive dynamics for other SSBs and variants across various systems.

Keywords: Competition; Exchange; FRET; Replacement; SSB; Single molecule; Transfer; ssDNA.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA, Single-Stranded / metabolism*
  • DNA-Binding Proteins / metabolism*
  • Escherichia coli Proteins / metabolism*
  • Fluorescence Resonance Energy Transfer / instrumentation
  • Fluorescence Resonance Energy Transfer / methods*
  • Image Processing, Computer-Assisted / instrumentation
  • Image Processing, Computer-Assisted / methods
  • Intravital Microscopy / instrumentation
  • Intravital Microscopy / methods*
  • Microscopy, Fluorescence / instrumentation
  • Microscopy, Fluorescence / methods
  • Protein Binding
  • Recombinational DNA Repair
  • Single Molecule Imaging / instrumentation
  • Single Molecule Imaging / methods*
  • Software
  • Video Recording / instrumentation
  • Video Recording / methods

Substances

  • DNA, Single-Stranded
  • DNA-Binding Proteins
  • Escherichia coli Proteins
  • SSB protein, E coli