Endonuclease activity of Escherichia coli DNA helicase I directed against the transfer origin of the F factor.
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.
Reygers
U
U
Max-Planck-Institut für Medizinische Forschung, Heidelberg, FRG.
Wessel
R
R
Müller
H
H
Hoffmann-Berling
H
H
D001483:Base Sequence; D002678:Chimera; D004265:DNA Helicases / Q000378:metabolism*; D004269:DNA, Bacterial; D004587:Electrophoresis, Agar Gel; D004720:Endonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005144:F Factor; D008969:Molecular Sequence Data; D015183:Restriction Mapping
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D004720:Endonucleases; C027093:TraI protein, E coli; D004265:DNA Helicases