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Fluorescence‐detected interactions of oligonucleotides in RecA complexes
Author(s) -
Wittung Pernilla,
Funk Mario,
Jernström Bengt,
Nordén Bengt,
Takahashi Masayuki
Publication year - 1995
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(95)00600-e
Subject(s) - oligonucleotide , chemistry , dna , guanine , fluorescence , protein filament , quenching (fluorescence) , chromophore , biophysics , pyrene , fluorescence anisotropy , biochemistry , stereochemistry , nucleotide , photochemistry , biology , physics , organic chemistry , quantum mechanics , membrane , gene
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(CTACTAAACAT TACAAATCATCC) 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 ATPγS, 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.