Evidence for ATP binding and double-stranded DNA binding by Escherichia coli RecF protein.
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.
Madiraju
M V
MV
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Clark
A J
AJ
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000345:Affinity Labels / Q000378:metabolism; D001386:Azides / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism* / Q000528:radiation effects; D001667:Binding, Competitive; D003432:Cross-Linking Reagents; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism* / Q000528:radiation effects; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D014466:Ultraviolet Rays
D000345:Affinity Labels; D001386:Azides; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C489340:recF protein, E coli; C022571:adenosine 5'-O-(3-thiotriphosphate); C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; C066218:recF protein, Bacteria