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

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.

Biochemical Characterization of Human Tyrosyl-DNA Phosphodiesterase 2 (TDP2/TTRAP)

“…Our study underlines the differences between TDP2 and TDP1, as TDP1 functions without metal cofactor and utilizes a transient covalent intermediate (13,44). Together with the prior finding that TDP2 can also process 3Ј-tyrosyl substrates, albeit with much lower affinity (12,29), our results suggest the broad range of activity of TDP2 and its preferential action at abortive Top2 and possibly Top3 cleavage complexes. ID 3FZI).…”

“…Nucleases remove a stretch of DNA containing the trapped topoisomerase, whereas TDP2 can specifically cleave the phosphotyrosyl linkage initially formed during Top2-mediated cleavage of DNA. Our data suggest that the 5Ј-tyrosyl-DNA phosphodiesterase activity of TDP2 is robust under various conditions including broad pH range and low Mg 2ϩ or Mn 2ϩ concentrations (29). The recently resolved crystal structure of trapped Top2 has shown that a single etoposide molecule intercalated at the cleavage sites of Top2 dislodges the two cleaved DNA ends, thereby disfavoring religation (35).…”

“…Mg 2ϩ concentration as low as 0.1 mM was sufficient for efficient cleavage of ssY19 substrate (Fig. 4C) (29).…”

Structural basis for recognition of 5′-phosphotyrosine adducts by Tdp2

“…2c–e, bottom panels). The conserved active site architecture indicates that TDP2 employs a divalent metal-dependent catalytic mechanism similarly to other members of this family 9,24 .…”

Tyrosyl-DNA-phosphodiesterases (TDP1 and TDP2)

“…Unlike TDP1, TDP2 requires divalent metals but does not form a transient covalent catalytic intermediate (Table 3) [129,134,138]. Mg 2+ , Mn 2+ , Co 2+ are much more efficient than Ca 2+ or Zn 2+ ) for catalysis [134].…”