Characterization of magnesium requirement of human 5'-tyrosyl DNA phosphodiesterase mediated reaction

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 Mg ²⁺ 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 Mg ²⁺/Mn ²⁺, and that neither Zn ²⁺ or nor Ca ²⁺ can activate hTDP2. Mg ²⁺ also controls 3'-phosphotyrosyl activity of TDP2. In MCF-7 cell extracts and de-yolked zebrafish embryo extracts, Mg ²⁺ controlled 5'-phosphotyrosyl activity. This study also showed that there is an optimal Mg ²⁺ concentration above which it is inhibitory for hTDP2 activity. Conclusion: These results altogether reveal the optimal Mg ²⁺ requirement in hTDP2 mediated reaction.