CHARACTERIZATION OF THE REACTION-PRODUCT OF THE ORIT NICKING REACTIONCATALYZED BY ESCHERICHIA-COLI DNA HELICASE-I

DNA helicase I, encoded on the Escherichia coli F plasmid, catalyzes a site- and strand-specific nicking reaction within the F plasmid origi n of transfer (oriT) to initiate conjugative DNA strand transfer. The product of the nicking reaction contains a single phosphodiester bond interruption as determined by single-nucleotide resolution mapping of both sides of the nick site. This analysis has demonstrated that the n ick is located at precisely the same site previously shown to be nicke d in vivo (T. L. Thompson, M. B. Centola, and R. C. Deonier, J. Mol. B iol. 207:505-512, 1989). In addition, studies with two oriT point muta nts have confirmed the specificity of the in vitro reaction. Character ization of the nicked DNA product has revealed a modified 5' end and a 3' OH available for extension by E. coli DNA polymerase I. Precipitat ion of nicked DNA with cold KCl in the presence of sodium dodecyl sulf ate suggests the existence of protein covalently attached to the nicke d DNA molecule. The covalent nature of this interaction has been direc tly demonstrated by transfer of radiolabeled phosphate from DNA to pro tein. On the basis of these results, we propose that helicase I become s covalently bound to the 5' end of the nicked DNA strand as part of t he reaction mechanism for phosphodiester bond cleavage. A model is pre sented to suggest how helicase I could nick the F plasmid at oriT and subsequently unwind the duplex DNA to provide single-stranded DNA for strand transfer during bacterial conjugation.