Visualization of RecA filaments and DNA by fluorescence microscopy

J Biochem. 2007 Feb;141(2):147-56. doi: 10.1093/jb/mvm033. Epub 2007 Jan 3.


We have developed two experimental methods for observing Escherichia coli RecA-DNA filament under a fluorescence microscope. First, RecA-DNA filaments were visualized by immunofluorescence staining with anti-RecA monoclonal antibody. Although the detailed filament structures below submicron scale were unable to be measured accurately due to optical resolution limit, this method has an advantage to analyse a large number of RecA-DNA filaments in a single experiment. Thus, it provides a reliable statistical distribution of the filament morphology. Moreover, not only RecA filament, but also naked DNA region was visualized separately in combination with immunofluorescence staining using anti-DNA monoclonal antibody. Second, by using cysteine derivative RecA protein, RecA-DNA filament was directly labelled by fluorescent reagent, and was able to observe directly under a fluorescence microscope with its enzymatic activity maintained. We showed that the RecA-DNA filament disassembled in the direction from 5' to 3' of ssDNA as dATP hydrolysis proceeded.

MeSH terms

  • DNA / analysis*
  • DNA, Single-Stranded / analysis
  • Deoxyadenine Nucleotides / analysis
  • Escherichia coli / chemistry*
  • Escherichia coli Proteins / analysis*
  • Fluorescent Antibody Technique
  • Microscopy, Fluorescence*
  • Rec A Recombinases / analysis*


  • DNA, Single-Stranded
  • Deoxyadenine Nucleotides
  • Escherichia coli Proteins
  • DNA
  • Rec A Recombinases