Binding of RecA protein to single-stranded nucleic acids: spectroscopic studies using fluorescent polynucleotides.
Binding of the recA gene product from Escherichia coli to single-stranded polynucleotides has been investigated using poly(dA) that have been modified by chloroacetaldehyde to yield fluorescent 1,N6-ethenoadenine (epsilon A) bases. A strong enhancement of the fluorescent quantum yield of poly(d epsilon A) is induced upon RecA protein binding. A 4-fold increase is observed in the absence of ATP or ATP gamma S and a 7-fold increase in the presence of either nucleoside triphosphate. RecA protein can bind to poly(d epsilon A) in the absence of both Mg2+ ions and ATP (or ATP gamma S) but Mg2+ ions are required to observe RecA protein binding in the presence of ATP (or ATP gamma S) at pH 7.5. ATP binding to the RecA-poly(d epsilon A) complex induces a dissociation of RecA from the polynucleotide followed by re-binding of [RecA-ATP-Mg2+] ternary complex. Whereas ATP-induced dissociation of RecA-poly(d epsilon A) complexes is a fast process, the subsequent binding reaction of [RecA-ATP-Mg2+] is slow. A model is proposed whereby [RecA-ATP-Mg2+] binding to poly(d epsilon A) involves slow nucleation and elongation processes along the polynucleotide backbone. The nucleation reaction is shown to involve at least a trimer or a tetramer. Polymerization of the [RecA-ATP-Mg2+] ternary complex stops when the polynucleotide is entirely covered with 6 +/- 1 nucleotides per RecA monomer. ATP hydrolysis then induces a release of RecA-ADP complexes from the polynucleotide template.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D004247:DNA / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D011061:Poly A / Q000378:metabolism*; D011485:Protein Binding; D011938:Rec A Recombinases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013950:Thymus Gland
D005456:Fluorescent Dyes; C040459:poly(d-epsilon A); D011061:Poly A; D000255:Adenosine Triphosphate; D004247:DNA; D011938:Rec A Recombinases; D008274:Magnesium