PMID: 8612607Mar 1, 1996Paper

Purification and characterization of human topoisomerase I mutants

European Journal of Biochemistry
A D Jensen, J Q Svejstrup

Abstract

A system for rapid purification and characterization of eukaryotic topoisomerase-I mutants has been developed. The system utilizes six-histidine tagging of human topoisomerase I expressed in Saccharomyces cerevisiae to enable purification by nickel-affinity chromatography. Virtually homogenous mutant proteins are then tested for their ability to relax supercoiled DNA plasmids and their capacity for binding, cleaving and religating short defined DNA substrates. Relaxation-deficient mutants were obtained by site-directed mutagenesis of selected highly conserved amino acids. The mutants Tyr723Phe (active site mutation), Arg488Gln and Lys532Glu were inert in relaxation of DNA, whereas Lys720Glu showed a 50-fold reduction in specific relaxation activity. Accordingly, only Lys720Glu showed low, but detectable cleavage activity on suicide DNA substrates, uncoupling the cleavage and religation events of topoisomerase I. The relative religation efficiency of Lys720Glu comparable to that of wild-type topoisomerase I, indicating that Lys720 is involved in interactions important for normal DNA cleavage, but not for the religation reaction. All mutants could be cross linked by ultraviolet light to bromo-dUTP-substituted DNA oligonucleotides...Continue Reading

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Citations

Mar 19, 2004·Bioorganic & Medicinal Chemistry·Craig J ThomasSidney M Hecht
Jan 27, 2004·Journal of Molecular Biology·Rikke From FrøhlichBirgitta Ruth Knudsen
Sep 28, 1998·Biochimica Et Biophysica Acta·R J ReidM A Bjornsti
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Mar 5, 2010·Proceedings. Biological Sciences·Carlos García-EstradaRafael Balaña-Fouce
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Jul 21, 2010·Bioorganicheskaia khimiia·D V Bugreev, G A Nevinskiĭ
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Sep 28, 1998·Biochimica Et Biophysica Acta·S Shuman
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Jun 8, 2002·Biochemical and Biophysical Research Communications·Hidehiro TakahashiTakeshi Kurata
Mar 21, 1998·Science·L StewartJ J Champoux

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