Escherichia coli DNA helicase I. Characterization of the protein and of its DNA-binding properties.
Gene traI of the Escherichia coli F sex factor which encodes DNA helicase I was subcloned in a lambda pL-based plasmid vector and expressed in a background of pL non-repressing cells. Neither the non-repressed pL promoter nor the production of a high level of functional helicase I are toxic. Enzyme purified from this source was studied in the electron microscope. The results show that helicase I binds cooperatively to single-stranded DNA. DNA covered with the helicase appears in fixed, negatively stained specimens as a smooth-contoured filament with a diameter of 12.5 +/- 0.4 nm and an axial periodicity of 7.0 +/- 0.2 nm. In unfixed specimens, discrete particles with axes of 12.7 +/- 0.5 nm and 7.2 +/- 0.5 nm are visible. They are consistent in size with helicase I monomers (Mr 180,000) suggesting that the molecule is almost isometric, despite a frictional ratio of 1.71 calculated from its diffusion coefficient. Helicase I free of DNA appears as aggregates. For comparison, a truncated traI, lacking coding for the amino-terminus of the product, was cloned by fusing it to an MS2 replicase gene fragment. The chimeric gene product (named helicase I del29) retains strand-separating activity although it fails to show cooperative DNA binding behavior. Judged from the length of the helicase-I-specific sequence of this polypeptide, traI is located 1.3 kb nearer to the distal end of the F transfer operon compared to the position proposed in a previous genetic map. The revised location of traI has implications for understanding distal functions of the transfer operon.
Benz
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I
Max-Planck-Institut für medizinische Forschunge, Abteilung Molekulare Biologie, Heidelberg, Federal Republic of Germany.
Müller
H
H
D001665:Binding Sites; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003090:Coliphages / Q000378:metabolism; D004265:DNA Helicases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism / Q000648:ultrastructure; D004279:DNA, Viral / Q000378:metabolism / Q000648:ultrastructure; D017886:Durapatite; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005144:F Factor; D005798:Genes, Bacterial; D006882:Hydroxyapatites; D008854:Microscopy, Electron; D011485:Protein Binding; D011487:Protein Conformation; D015183:Restriction Mapping
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D029968:Escherichia coli Proteins; D006882:Hydroxyapatites; D017886:Durapatite; C027093:TraI protein, E coli; D004265:DNA Helicases