Cofactor-induced orientation of the DNA bases in single-stranded DNA complexed with RecA protein. A fluorescence anisotropy and time-decay study.
The structure of the RecA-single-stranded DNA complex was investigated by studying the fluorescence emission of poly(deoxy-1,N6-ethenoadenylic acid (poly(d epsilon A)), a fluorescent derivative of poly(dA), under various viscosity conditions. The fluorescence intensity and average lifetime of poly(d epsilon A) are much smaller than those of nonpolymerized monoethenonucleotides (1,N6-ethenoadenosine 5'-triphosphate and 1,N6-ethenoadenine deoxyribose 5'-monophosphate) at low viscosity and reflect intramolecular base-base collisions in the polymer. They considerably increased upon RecA binding, both in the presence and absence of cofactor ATP or adenosine 5'-O-(3-thiotriphosphate). This increase, as well as the increase in fluorescence anisotropy upon RecA binding, was very similar to that which resulted from sucrose addition to free poly(d epsilon A). These observations point to a decrease in the mobility of DNA bases upon RecA binding. In the presence of cofactor, the fluorescence features became independent of viscosity. This strongly suggests the absence of base motion of significant amplitude on the time scale of the fluorescence lifetime (about 10 ns). In the absence of cofactor, however, these features remained sensitive to viscosity, implying residual local motions of the bases. Such cofactor-dependent rigid attachment of DNA bases to stiff phosphate backbone could facilitate the search for homology between two DNA molecules during recombination.
Chabbert
M
M
Faculté de Pharmacie, Université Louis Pasteur, Illkirch, France.
Lami
H
H
Takahashi
M
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005454:Fluorescence Polarization; D009690:Nucleic Acid Conformation; D011061:Poly A / Q000378:metabolism; D011487:Protein Conformation; D011938:Rec A Recombinases / Q000378:metabolism*; D013395:Sucrose
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D011061:Poly A; C015465:poly(dA); D013395:Sucrose; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases