Large scale purification and characterization of TraI endonuclease encoded by sex factor plasmid R100.
The TraI protein encoded by plasmid R100 was purified in a large scale by monitoring the strand- and site-specific nicking activity at the origin of transfer, oriT. The N-terminal amino acid sequence of the purified protein was identical to that deduced from the DNA sequence of an open reading frame encoding TraI. The TraI protein is a DNA helicase which is highly processive and unwinds DNA in the 5' to 3' direction. The Stokes radius and the sedimentation coefficient for the TraI protein in 200 mM NaCl indicate that the protein is a rod-shaped monomer, whose native molecular weight is 186,000. Chemical cross-linking analysis revealed that there exist more dimers of TraI under the low salt conditions, under which both nicking and unwinding reactions catalyzed by TraI are the most efficient, indicating that the TraI protein is functionally active in a dimer form. TraI hardly introduced a nick into the linearized plasmid DNA and only slightly into the relaxed closed circular DNA, indicating that TraI requires superhelical structure of substrate DNA for the nicking reaction. Deletion analysis in the oriT region revealed that a particular region of 54 base pairs containing oriT is required for the nicking reaction.
Fukuda
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H
Institute of Molecular and Cellular Biosciences, University of Tokyo, Japan.
Ohtsubo
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E
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D003227:Conjugation, Genetic; D004265:DNA Helicases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D017931:DNA Primers; D004278:DNA, Superhelical / Q000737:chemistry; D004720:Endonucleases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011815:R Factors; D018741:Replication Origin / Q000235:genetics
D017931:DNA Primers; D004278:DNA, Superhelical; D029968:Escherichia coli Proteins; D004720:Endonucleases; C027093:TraI protein, E coli; D004265:DNA Helicases