Fluorescence-detected interactions of oligonucleotides in RecA complexes
Journal article, 1995
A technique has been developed to probe directly RecA-DNA interactions by the use of the fluorescent chromophore, (+)anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), covalently attached to DNA, The 24-mer oligonucleotide 5'-d(CTACTAAACATGTACAAATCATCC) was specifically modified on the exocyclic nitrogen of the central guanine, to yield a trans-adduct. Upon interaction of the modified oligonucleotide with RecA we find an increase in BPDE fluorescence and a rather high fluorescence anisotropy, suggesting a restricted motion of the BPDE-oligonucleotide in the protein filament. In the presence of the cofactor ATP gamma S, binding of two oligonuclotides, identical or complementary in sequence, in the RecA filament is possible, The RecA-DNA complex is, however, more stable when the sequences are complementary; in addition, a shift in the BPDE emission peaks is observed, In the presence of ATP (and an ATP regeneration system), the RecA-DNA interaction between two complementary oligonucleotides is changed, and we now find protein-mediated renaturation to occur.
escherichia-coli
solution
stoichiometry
spectroscopy
protein
mechanism
genetic-recombination
linear dichroism
reca
coordination
benzo(a)pyrenediolepoxide
conformation
dna complexes
recombination
fluorescence
renaturation
Author
Pernilla Wittung Stafshede
Department of Physical Chemistry
M. Funk
B. Jernstrom
Bengt Nordén
Department of Physical Chemistry
M. Takahashi
FEBS Letters
0014-5793 (ISSN)
Vol. 368 1 64-68Subject Categories (SSIF 2011)
Biochemistry and Molecular Biology
DOI
10.1016/0014-5793(95)00600-E