Evidence for ATP binding and double-stranded DNA binding by Escherichia coli RecF protein

Abstract

RecF protein is one of the important proteins involved in DNA recombination and repair. RecF protein has been shown to bind single-stranded DNA (ssDNA) in the absence of ATP (T. J. Griffin IV and R. D. Kolodner, J. Bacteriol. 172:6291-6299, 1990; M. V. V. S. Madiraju and A. J. Clark, Nucleic Acids Res. 19:6295-6300, 1991). In the present study, using 8-azido-ATP, a photo-affinity analog of ATP, we show that RecF protein binds ATP and that the binding is specific in the presence of DNA. 8-Azido-ATP photo-cross-linking is stimulated in the presence of DNA (both ssDNA and double-stranded DNA [dsDNA]), suggesting that DNA enhances the affinity of RecF protein for ATP. These data suggest that RecF protein possesses independent ATP- and DNA-binding sites. Further, we find that stable RecF protein-dsDNA complexes are obtained in the presence of ATP or ATP-gamma-S [adenosine-5'-O-(3-thio-triphosphate)]. No other nucleoside triphosphates served as necessary cofactors for dsDNA binding, indicating that RecF is an ATP-dependent dsDNA-binding protein. Since a mutation in a putative phosphate-binding motif of RecF protein results in a recF mutant phenotype (S. J. Sandler, B. Chackerian, J. T. Li, and A. J. Clark, Nucleic Acids Res. 20:839-845, 1992), we suggest on the basis of our data that the interactions of RecF protein with ATP, with dsDNA, or with both are physiologically important for understanding RecF protein function in vivo.

RecR-mediated Modulation of RecF Dimer Specificity for Single- and Double-stranded DNA

“…All known ABC-type ATPases function as oligomeric complexes in which a sequence of inter-and intra-molecular interactions is triggered by the ATP-dependent dimerization and the dimer-dependent ATP hydrolysis (36 -39). RecF is also an ATP-dependent DNA-binding protein and a weak DNA-dependent ATPase (11,40). RecF forms an ATP-dependent dimer and all three conserved motifs (Walker A, Walker B, and "signature") of RecF are important for ATPdependent dimerization, ATP hydrolysis, and functional resistance to DNA damage (35).…”

“…S5). The E. coli RecF binds plasmid DNA in the presence of ATP␥S (11,40), while in our experiments ATP␥S did not support stable interaction between the D. radiodurans RecF and short oligonucleotides in the absence of RecR. Such a difference may arise from slightly shifted equilibrium of RecF dimerization caused by either different experimental conditions, different substrates (short oligonucleotides versus plasmid DNA where binding of multiple dimers to the same DNA molecule may play a stabilizing effect), or minor differences in dimerization constants of E. coli and D. radiodurans RecF proteins.…”

Interactions of RecF protein with RecO, RecR, and single-stranded DNA binding proteins reveal roles for the RecF–RecO–RecR complex in DNA repair and recombination

“…The RecF protein binds ATP, and binding of RecF to ATP is essential for its binding to dsDNA (22) and for it to exhibit preferential binding to gapped (g) DNA (24). Hence the effect of ATP on the interaction of RecF with RecO was examined using the immunoprecipitation techniques.…”

“…In this study, we have evaluated the interactions of RecF with RecO, RecR, and Ssb proteins individually and together. Since RecF binds ATP (22), the effect of ATP on these interactions has also been examined. Our results indicate that RecF protein physically interacts with RecO and with RecORecR complex.…”

Identification of the RecR Toprim Domain as the Binding Site for both RecF and RecO

“…recombinational DNA repair at replication forks (17,18). The RecF protein itself binds DNA and has a weak ATPase activity (19), both of which are enhanced by RecR binding (20,21). Furthermore, specific loading of the RecA protein onto the dsDNA-ssDNA junction is mediated by the RecF, RecO, and RecR proteins acting in concert, with no interaction between RecF and RecO detected (4).…”