Binding of RecA protein to single‐stranded nucleic acids: spectroscopic studies using fluorescent polynucleotides

• Published in: The EMBO journal Vol. 2; no. 12; pp. 2247 - 2251
• Main Authors: Cazenave, C., Toulmé, J.J., Hélène, C.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group 01.01.1983
• Subjects: Adenosine Triphosphate - metabolism; Animals; Biological and medical sciences; Cattle; DNA - metabolism; Escherichia coli - genetics; Fluorescent Dyes - metabolism; Fundamental and applied biological sciences. Psychology; Genic rearrangement. Recombination. Transposable element; Kinetics; Magnesium - metabolism; Molecular and cellular biology; Molecular genetics; Poly A - metabolism; Protein Binding; Rec A Recombinases - metabolism; Spectrometry, Fluorescence; Thymus Gland; Proteins; Recombination; Polynucleotide; DNA; Escherichia coli; Fluorescence; Polymerization; Bacteria; Molecular interaction; Models; Repair; Mechanism
• ISSN: 0261-4189; 1460-2075
• DOI: 10.1002/j.1460-2075.1983.tb01730.x

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.