Post-translational modifications in DNA topoisomerase 2\u03b1 highlight the role of a eukaryote-specific residue in the ATPase domain

Claire Bedez,Eduardo Howard,Claire Batisse,Marc Ruff,Christophe Lotz,Karine Pradeau-Aubreton,Roland H Stote,Arnaud Vanden Broeck,Valérie Lamour

doi:10.1038/s41598-018-27606-8

Abstract

Type 2 DNA topoisomerases (Top2) are critical components of key protein complexes involved in DNA replication, chromosome condensation and segregation, as well as gene transcription. The Top2 were found to be the main targets of anticancer agents, leading to intensive efforts to understand their functional and physiological role as well as their molecular structure. Post-translational modifications have been reported to influence Top2 enzyme activities in particular those of the mammalian Top2α isoform. In this study, we identified phosphorylation, and for the first time, acetylation sites in the human Top2α isoform produced in eukaryotic expression systems. Structural analysis revealed that acetylation sites are clustered on the catalytic domains of the homodimer while phosphorylation sites are located in the C-terminal domain responsible for nuclear localization. Biochemical analysis of the eukaryotic-specific K168 residue in the ATPase domain shows that acetylation affects a key position regulating ATP hydrolysis through the modulation of dimerization. Our findings suggest that acetylation of specific sites involved in the allosteric regulation of human Top2 may provide a mechanism for modulation of its catalytic activity.

Highlights

Type 2 DNA topoisomerases (Top2) are critical components of key protein complexes involved in DNA replication, chromosome condensation and segregation, as well as gene transcription
Our study shows that post-translational modifications may impact conserved residues of Top2α and variable regions It suggests that the post-translational modifications of the recombinant enzymes should be taken into account when analyzing their functional properties and sensitivity to inhibitors
In order to characterize the post-translational modification state of our samples, we identified the phosphorylation and acetylation sites of the HsTop2α overexpressed in the two eukaryotic expression systems

Summary

Introduction

Type 2 DNA topoisomerases (Top2) are critical components of key protein complexes involved in DNA replication, chromosome condensation and segregation, as well as gene transcription. Phosphorylation sites are the best characterized with a number of studies showing their impact on the regulation of Top[2] enzyme function, nuclear localization and control of cell cycle checkpoint[16,17,18,19,20,21,22]. Both isoforms of human Top[2] are phosphorylated throughout the cell cycle and some positions are hyperphosphorylated during G2/M21,23–25. Hyperphosphorylation of the α isoform has been reported in etoposide resistant cells, while other studies associated a hypophosphorylation of the enzyme after doxorubicin treatment[29,31,32]

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Conclusion