PMID:1651938
| Citation |
Matson, SW and Morton, BS (1991) Escherichia coli DNA helicase I catalyzes a site- and strand-specific nicking reaction at the F plasmid oriT. J. Biol. Chem. 266:16232-7 |
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| Abstract |
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. |
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| Keywords |
Base Sequence; Catalysis; Conjugation, Genetic; Cross-Linking Reagents; DNA Helicases/metabolism; DNA Replication; DNA, Bacterial/metabolism; DNA, Superhelical/metabolism; Electrophoresis, Agar Gel; Endonucleases/metabolism; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins; F Factor; Molecular Sequence Data |
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