Nucleic Acids Research, Vol 26, Issue 2 650-654, Copyright © 1998 by Oxford University Press
EN Zaitsev and SC Kowalczykowski
We have developed a new assay to characterize the double-stranded DNA
(dsDNA) binding properties of RecA protein. This assay is based on
measurement of changes in the fluorescence of a 4',6-diamidino-2-
phenylindole (DAPI)-dsDNA complex upon RecA protein binding. The binding of
RecA protein to a complex of DAPI and dsDNA results in displacement of the
bound DAPI, producing a decrease in the observed fluorescence. DAPI
displacement is dependent on both RecA protein and ATP; dATP and, to a
lesser extent, UTP and dCTP also support the DAPI displacement reaction,
but dGTP, GTP, dITP and TTP do not. Binding stoichiometry for the RecA
protein-dsDNA complex measured by DAPI displacement is 3 bp per RecA
protein monomer in the presence of ATP. These results, taken together with
data for mutant RecA proteins, suggest that this DAPI displacement assay
monitors formation of the high affinity DNA binding state of RecA protein.
Since this state of RecA protein defines the form of the nucleoprotein
filament that is active in DNA strand exchange, these findings raise the
possibility that the RecA protein-dsDNA filament may possess a homologous
pairing capacity.
ARTICLES
Binding of double-stranded DNA by Escherichia coli RecA protein monitored by a fluorescent dye displacement assay
Division of Biological Sciences, Sections of Microbiology and Molecular and Cell Biology, University of California, Davis, CA 95616-8665, USA.
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