Characterization of an ATP-dependent type I DNA ligase from Arabidopsis thaliana |
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Authors: | Wu You-Qiang Hohn Barbara Ziemienowicz Alicja |
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Institution: | (1) Friedrich Miescher-Institute, P.O. Box 2543, 4002 Basel, Switzerland;(2) Present address: Plant Protection and Biotechnology Laboratory, Dept. of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk, ul. Kladki 24, 80-822 Gdansk, Poland |
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Abstract: | Here we report the purification and biochemical characterization of recombinant Arabidopsis thaliana DNA ligase I. We show that this ligase requires ATP as a source for adenylation. The calculated K
m ATP] for ligation is 3 M. This enzyme is able to ligate nicks in oligo(dT)/poly(dA) and oligo(rA)/poly(dT) substrates, but not in oligo(dT)/poly(rA) substrates. Double-stranded DNAs with cohesive or blunt ends are also good substrates for the ligase. These biochemical features of the purified enzyme show the characteristics typical of a type I DNA ligase. Furthermore, this DNA ligase is able to perform the reverse reaction (relaxation of supercoiled DNA) in an AMP-dependent and PPi-stimulated manner. |
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Keywords: | Arabidopsis thaliana DNA ligase intein fusion ligation relaxation |
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