1. Characterization of magnesium requirement of human 5'-tyrosyl DNA phosphodiesterase mediated reaction.
- Author
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Adhikari S, Karmahapatra SK, Karve TM, Bandyopadhyay S, Woodrick J, Manthena PV, Glasgow E, Byers S, Saha T, and Uren A
- Subjects
- Animals, Calcium metabolism, Cell Extracts chemistry, DNA metabolism, DNA-Binding Proteins, Electrophoresis, Polyacrylamide Gel, Embryo, Nonmammalian embryology, Enzyme Activation, Escherichia coli genetics, Fish Proteins isolation & purification, Humans, MCF-7 Cells, Nuclear Proteins genetics, Nuclear Proteins isolation & purification, Oligonucleotides metabolism, Phosphoric Diester Hydrolases, Recombinant Proteins genetics, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Tissue Extracts chemistry, Transcription Factors genetics, Transcription Factors isolation & purification, Zebrafish embryology, Zinc metabolism, Embryo, Nonmammalian enzymology, Fish Proteins metabolism, Magnesium metabolism, Manganese metabolism, Nuclear Proteins metabolism, Transcription Factors metabolism, Zebrafish metabolism
- Abstract
Background: Topo-poisons can produce an enzyme-DNA complex linked by a 3'- or 5'-phosphotyrosyl covalent bond. 3'-phosphotyrosyl bonds can be repaired by tyrosyl DNA phosphodiesterase-1 (TDP1), an enzyme known for years, but a complementary human enzyme 5'-tyrosyl DNA phosphodiesterase (hTDP2) that cleaves 5'-phosphotyrosyl bonds has been reported only recently. Although hTDP2 possesses both 3'- and 5'- tyrosyl DNA phosphodiesterase activity, the role of Mg2+ in its activity was not studied in sufficient details., Results: In this study we showed that purified hTDP2 does not exhibit any 5'-phosphotyrosyl phosphodiesterase activity in the absence of Mg2+/Mn2+, and that neither Zn2+ or nor Ca2+ can activate hTDP2. Mg2+ also controls 3'-phosphotyrosyl activity of TDP2. In MCF-7 cell extracts and de-yolked zebrafish embryo extracts, Mg2+ controlled 5'-phosphotyrosyl activity. This study also showed that there is an optimal Mg2+ concentration above which it is inhibitory for hTDP2 activity., Conclusion: These results altogether reveal the optimal Mg2+ requirement in hTDP2 mediated reaction.
- Published
- 2012
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