The simultaneous binding of two double-stranded DNA molecules by Escherichia coli RecA protein

Eugene N. Zaitsev, Stephen C. Kowalczykowski

Research output: Contribution to journalArticlepeer-review

17 Scopus citations


We have characterized the double-stranded DNA (dsDNA) binding properties of RecA protein, using an assay based on changes in the fluorescence of 4',6-diamidino-2-phenylindole (DAPI)-dsDNA complexes. Here we use fluorescence, nitrocellulose filter-binding, and DNase I-sensitivity assays to demonstrate the binding of two duplex DNA molecules by the RecA protein filament. We previously established that the binding stoichiometry for the RecA protein-dsDNA complex is three base-pairs per RecA protein monomer, in the presence of ATP. In the presence of ATPγS, however, the binding stoichiometry depends on the MgCl2 concentration. The stoichiometry is 3 bp per monomer at low MgCl2 concentrations, but changes to 6 bp per monomer at higher MgCl2 concentrations, with the transition occurring at approximately 5 mM MgCl2. Above this MgCl2 concentration, the dsDNA within the RecA nucleoprotein complex becomes uncharacteristically sensitive to DNase I digestion. For these reasons we suggest that, at the elevated MgCl2 conditions, the RecA-dsDNA nucleoprotein filament can bind a second equivalent of dsDNA. These results demonstrate that RecA protein has the capacity to bind two dsDNA molecules, and they suggest that RecA or RecA-like proteins may effect homologous recognition between intact DNA duplexes.

Original languageEnglish (US)
Pages (from-to)21-31
Number of pages11
JournalJournal of Molecular Biology
Issue number1
StatePublished - Mar 19 1999


  • DNA binding
  • DNA strand exchange
  • Homologous pairing
  • RecA protein
  • Recombination

ASJC Scopus subject areas

  • Virology


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