Purification and characterisation of a DNA helicase, dheI I, from Drosophila melanogaster embryos.
We have purified a DNA helicase (dhel l) from early Drosophila embryos. dhel l co-purifies with the single-stranded DNA binding protein dRP-A over two purification steps, however, the proteins can be separated by their different native molecular weight, with dhel l activity co-sedimenting with a polypeptide of approximately 200 kDa and a sedimentation coefficient of 8.6 S. The enzyme needs ATP hydrolysis and divalent cations for displacement activity. It is very salt sensitive, having a Mg2+ optimum of 0.5 mM and being inhibited by NaCl concentration > 10 mM. Dhel l moves 5'-->3' on the DNA strand to which it is bound. Unwinding activity decreases with increasing length of the double-stranded region suggesting a distributive mode of action. However, addition of dRP-A to the displacement reaction stimulates the activity on substrates with >300 nucleotides double-stranded region suggesting a specific interaction between these two proteins.
Thömmes
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Marie Curie Research Institute, Oxted, Surrey, UK.
Marton
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Cotterill
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D000251:Adenosine Triphosphatases / Q000032:analysis; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases / Q000302:isolation & purification* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000196:embryology / Q000201:enzymology*; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D011485:Protein Binding; D051716:Replication Protein A
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D029968:Escherichia coli Proteins; D051716:Replication Protein A; C497166:RpA-70 protein, Drosophila; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases; C027093:TraI protein, E coli; D004265:DNA Helicases