Effect of heavy metal binding on the function of Escherichia coli topoisomerase I

Abstract:

[Background] Escherichia coli topoisomerase I (E. coli TopA) plays essential role in DNA replication, transcription, recombination and regulation of gene expression. It is active only if it is bound with zinc. However, it remains unknown if it could bind other metals, especially heavy metals. [Objective] This study is aimed to characterize the binding activity of E. coli TopA to some toxic heavy metals in the environment, and to verify the potential effect of heavy metals on the topoisomerase activity of E. coli TopA. [Methods] We expressed and purified E. coli TopA from E. coli cells grown in M9 minimal media supplemented with exogenous zinc, cobalt, nickel, cadmium, iron, mercury, arsenic, chromium, lead or copper separately. The metal contents of purified TopA proteins were determined by ICP-MS (Inductively coupled plasma mass spectrometry). By using E. coli TopA mutants in which the zinc-finger motifs were disrupted, the binding sites of these metals in TopA were identified. The in vitro DNA relaxation assay was conducted to study the topoisomerase activity of E. coli TopA bound with different metals. Finally, intrinsic fluorescences of E. coli TopA bound with different metals were also measured to discover potential conformation diversity among them. [Results] We found that E. coli TopA could maximally bind three atoms of cobalt, nickel or cadmium per protein monomer respectively, while could not bind mercury, arsenic, chromium, lead or copper. The binding sites of these three metals in TopA were just located in the zinc-finger motifs, in which one atom of metal iron was coordinated with each zinc-finger motif respectively. It also showed that the DNA topoisomerase activity of TopA remained active when bound with cobalt, nickel or cadmium. Moreover, intrinsic fluorescence measurement showed that the protein conformation of cobalt, nickel or cadmium bound TopA would be similar as that of zinc bound. [Conclusion] In light of the crucial role of DNA topoisomerase in maintenance of cell viability, these results indicate that the topoisomerase activity of TopA would not be disrupted by common heavy metals (no binding or binding without inhibition of enzyme activity), which could be considered as an evolutionary mechanism for E. coli in resistance to toxicity of heavy metals in environment.