PMID:1651233
| Citation |
Reygers, U, Wessel, R, Müller, H and Hoffmann-Berling, H (1991) Endonuclease activity of Escherichia coli DNA helicase I directed against the transfer origin of the F factor. EMBO J. 10:2689-94 |
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| Abstract |
DNA helicase I, the traI gene product of the Escherichia coli F factor, was shown to be associated with endonuclease activity specific for the transfer origin of the F plasmid, oriT. In the presence of Mg2+, the purified enzyme forms a complex, stable in the presence of sodium dodecylsulfate (SDS) with a negatively superhelical chimeric plasmid containing oriT. The enzyme nicks and, after this, apparently binds to the 5' nick terminus when this complex is heated in the presence of SDS and/or EDTA or treated with proteinase K. Dideoxy sequencing locates the nick site in the F DNA strand transferred during bacterial conjugation after nucleotide 138 clockwise of the mid-point of the BglII site at 66.7 kb of the F genetic map. A sequencing stop after nucleotide 137 of this strand (where oriT-nicking seems to occur in vivo) is possibly an artefact caused by helicase I protein attached to the 5' terminal nucleotide. Deletion in the amino-terminal part of the traI polypeptide abolishes the oriT-nicking activity while leaving the strand-separating activity intact. These results confirm the prediction from genetic studies that helicase I is bifunctional with site-specific endonuclease and strand-separating activities. |
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| Keywords |
Base Sequence; Chimera; DNA Helicases/metabolism; DNA, Bacterial; Electrophoresis, Agar Gel; Endonucleases/metabolism; Escherichia coli/enzymology; Escherichia coli Proteins; F Factor; Molecular Sequence Data; Restriction Mapping |
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