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

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 demonstrat e 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 ATP gamma S, 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 MgCl 2, concentrations, with the transition occurring at approximately 5 mM MgCl 2. Above this MgCl2 concentration, the dsDNA within the RecA nucleoprotein complex becomes uncharacteristically sensitive to DNase I digestion. For th ese reasons we suggest that, at the elevated MgCl2 conditions, the RecA-dsD NA nucleoprotein filament can bind a second equivalent of dsDNA. These resu lts demonstrate that RecA protein has the capacity to bind two dsDNA molecu les, and they suggest that RecA or RecA-like proteins may effect homologous recognition between intact DNA duplexes. (C) 1999 Academic Press.