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 pol...

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Bibliographic Details
Published in:Nucleic acids research 1995-11, Vol.23 (21), p.4443-4450
Main Authors: Thömmes, P, Marton, R F, Cotterill, S
Format: Article
Language:English
Subjects:
Adenosine Triphosphatases - analysis
Adenosine Triphosphate - metabolism
Animals
Base Sequence
Centrifugation, Density Gradient
Chromatography, Gel
DNA - metabolism
DNA Helicases - isolation & purification
DNA Helicases - metabolism
DNA-Binding Proteins - isolation & purification
Drosophila melanogaster
Drosophila melanogaster - embryology
Drosophila melanogaster - enzymology
Drosophila Proteins
Escherichia coli Proteins
Molecular Sequence Data
Protein Binding
Replication Protein A
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Summary: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.
ISSN:0305-1048
1362-4962