Type II topoisomerases are essential enzymes for solving DNA topological problems by passing one segment of DNA duplex through a transient double-strand break in a second segment. The reaction requires the enzyme to precisely control the DNA cleavage and gate opening coupled with ATP hydrolysis. Using pulse-alkylation mass spectrometry, we were able to monitor the solvent accessibilities around 13 cysteines distributed throughout human topoisomerase IIα by measuring the thiol reactivities with monobromobimane. Most of the measured reactivities are in accordance with the predicted ones based on a homology structural model generated from available crystal structures. However, these results reveal new information for both the residues not covered in the structural model and potential differences between the modeled and solution holoenzyme structures. Furthermore, based on the reactivity changes of several cysteines located at the N-gate and DNA-gate, we could monitor the movement of topoisomerase II in the presence of cofactors and detect differences in DNA-gate between two closed-clamp enzyme conformations locked by either AMPPNP or an anticancer drug, ICRF-193.
The article is published online in the Journal of Biological Chemistry and is free to access.