Escherichia coli DNA helicase I
- 1 April 1990
- journal article
- research article
- Published by Wiley in European Journal of Biochemistry
- Vol. 189 (2), 267-276
- https://doi.org/10.1111/j.1432-1033.1990.tb15486.x
Abstract
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 de129) 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.This publication has 30 references indexed in Scilit:
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