PMID:10074404
| Citation |
Zaitsev, EN and Kowalczykowski, SC (1999) The simultaneous binding of two double-stranded DNA molecules by Escherichia coli RecA protein. J. Mol. Biol. 287:21-31 |
|---|---|
| Abstract |
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 ATPgammaS, 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. |
| Links |
PubMed Online version:10.1006/jmbi.1998.2580 |
| Keywords |
Adenosine Triphosphate/analogs & derivatives; Adenosine Triphosphate/metabolism; Collodion; DNA, Bacterial/metabolism; Deoxyribonuclease I/metabolism; Escherichia coli; Ethidium; Indoles/metabolism; Magnesium Chloride/pharmacology; Models, Chemical; Protein Binding/drug effects; Rec A Recombinases/metabolism; Recombination, Genetic; Spectrometry, Fluorescence |
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