Escherichia coli DNA helicase I catalyzes a site- and strand-specific nicking reaction at the F plasmid oriT.
A site- and strand-specific nick, introduced in the F plasmid origin of transfer, initiates conjugal DNA transfer during bacterial conjugation. Recently, molecular genetic studies have suggested that DNA helicase I, which is known to be encoded on the F plasmid, may be involved in this nicking reaction (Traxler, B. A., and Minkley, E. G., Jr. (1988) J. Mol. Biol. 204, 205-209). We have demonstrated this site- and strand-specific nicking event using purified helicase I in an in vitro reaction. The nicking reaction requires a superhelical DNA substrate containing the F plasmid origin of transfer, Mg2+ and helicase I. The reaction is protein concentration-dependent but, under the conditions used, only 50-70% of the input DNA substrate is converted to the nicked species. Genetic data (Everett, R., and Willetts, N. (1980) J. Mol. Biol. 136, 129-150) have also suggested the involvement of a second F-encoded protein, the TraY protein, in the oriT nicking reaction. Unexpectedly, the in vitro nicking reaction does not require the product of the F plasmid traY gene. The implications of this result are discussed. The phosphodiester bond interrupted by helicase I has been shown to correspond exactly to the site nicked in vivo suggesting that helicase I is the site- and strand-specific nicking enzyme that initiates conjugal DNA transfer. Thus, helicase I is a bifunctional protein which catalyzes site- and strand-strand specific nicking of the F plasmid in addition to the previously characterized duplex DNA unwinding (helicase) reaction.
Matson
S W
SW
Department of Biology, University of North Carolina, Chapel Hill 27599.
Morton
B S
BS
D001483:Base Sequence; D002384:Catalysis; D003227:Conjugation, Genetic; D003432:Cross-Linking Reagents; D004265:DNA Helicases / Q000378:metabolism*; D004261:DNA Replication; D004269:DNA, Bacterial / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D004720:Endonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005144:F Factor; D008969:Molecular Sequence Data
D003432:Cross-Linking Reagents; D004269:DNA, Bacterial; D004278:DNA, Superhelical; D029968:Escherichia coli Proteins; D004720:Endonucleases; C027093:TraI protein, E coli; D004265:DNA Helicases