Single-molecule analysis of SSB dynamics on single-stranded DNA

Methods Mol Biol. 2012;922:85-100. doi: 10.1007/978-1-62703-032-8_5.

Abstract

SSB proteins bind to and control the accessibility of single-stranded (ss) DNA generated as a transient intermediate during a variety of cellular processes. For subsequent DNA processing, however, SSB needs to be removed and yield to other proteins while avoiding ssDNA exposure to nucleases. Using single-molecule two- and three-color fluorescence resonance energy transfer (FRET) and fluorescence-force spectroscopy, we recently showed that the SSB/DNA complex is a highly dynamic system and SSB functions as a sliding platform that migrates on ssDNA for recruiting other proteins in DNA repair, replication, and recombination. Here, we present the activity assays in detail for observing the transitions between different SSB binding modes and SSB diffusion on ssDNA in real time by using single-molecule FRET microscopy and for studying how mechanical forces regulate SSB-DNA interactions using fluorescence-force spectroscopy. These single-molecule approaches are generally applicable to many other protein-nucleic acid systems.

Publication types

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

MeSH terms

  • DNA, Single-Stranded / chemistry
  • DNA, Single-Stranded / metabolism*
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / metabolism*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Fluorescence Resonance Energy Transfer / methods*
  • Molecular Biology / methods
  • Spectrometry, Fluorescence / methods*

Substances

  • DNA, Single-Stranded
  • DNA-Binding Proteins
  • Escherichia coli Proteins