Abstract
BackgroundTopo-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.ResultsIn 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.ConclusionThese results altogether reveal the optimal Mg2+ requirement in hTDP2 mediated reaction.
Highlights
The topoisomerase II (TopII) family is an important class of topoisomerases whose activity consists of reaction cycles of DNA binding, DNA cleavage, DNA strand passage, and religation of the cleaved DNA
We demonstrate that absolutely no product was formed in the human TDP2 (hTDP2)-mediated reaction in the absence of Mg2+, even with a higher concentration of hTDP2 but there is an optimal Mg2+ concentration above which it is inhibitory for hTDP2 activity
The best fraction of hTDP2 protein was used, which is more than 95% pure electrophoretically (Figure 1)
Summary
The topoisomerase II (TopII) family is an important class of topoisomerases whose activity consists of reaction cycles of DNA binding, DNA cleavage, DNA strand passage, and religation of the cleaved DNA. The 5’-tyrosyl DNA phosphodiesterase activity of hTDP2 can enable the repair of TopII-induced double strand breaks (DSBs) without the need for nuclease activity, because it creates a “clean” DSB with 5’-phosphate termini and a 3’-hydroxyl group. These “clean” DSBs are religatable by DNA ligase, providing an opportunity for error free repair [9,10]. HTDP2 possesses both 3’- and 5’- tyrosyl DNA phosphodiesterase activity, the role of Mg2+ in its activity was not studied in sufficient details
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