PREFERENTIAL BINDING OF ESCHERICHIA-COLI RECF PROTEIN TO GAPPED DNA IN THE PRESENCE OF ADENOSINE (GAMMA-THIO) TRIPHOSPHATE

Escherichia coli RecF protein binds, but does not hydrolyze, ATP. To d etermine the role that ATP binding to RecF plays in RecF protein-media ted DNA binding, we have determined the interaction between RecF prote in and single-stranded (ss)DNA, double-stranded (ds)DNA, and dsDNA con taining ssDNA regions (gapped [g]DNA) either alone or in various combi nations both in the presence and in the absence of adenosine (gamma-th io) triphosphate, gamma-S-ATP, a nonhydrolyzable ATP analog. Protein-D NA complexes were analyzed by electrophoresis on agarose gels and visu alized by autoradiography. The type of protein-DNA complexes formed in the presence of gamma-S-ATP was different with each of the DNA substr ates and from those formed in the absence of gamma-S-ATP. Competition experiments with various combinations of DNA substrates indicated that RecF protein preferentially bound gDNA in the presence of gamma-S-ATP , and the order of preference of binding was gDNA > dsDNA > ssDNA. Sin ce gDNA has both ds- and ssDNA components, we suggest that the role fo r ATP in RecF protein-DNA interactions in vivo is to confer specificit y of binding to dsDNA-ssDNA junctions, which is necessary for catalyzi ng DNA repair and recombination.