Abstract
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 DNA cleavage and gate opening coupled with ATP hydrolysis. Using pulsed 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, on the basis of 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 the DNA gate between two closed clamp enzyme conformations locked by either 5'-adenylyl β,γ-imidodiphosphate or the anticancer drug ICRF-193.
Highlights
The catalytic cycle of Top2 has multiple steps of reactions with corresponding conformational changes
On the basis of the reactivity changes of several cysteines located at the N-terminal gate (N-gate) and DNA gate, we could monitor the movement of topoisomerase II in the presence of cofactors and detect differences in the DNA gate between two closed clamp enzyme conformations locked by either 5-adenylyl ,␥-imidodiphosphate or the anticancer drug ICRF-193
We have demonstrated that by using pulsed alkylation with mass spectrometric analysis, we were able to differentiate the levels of alkylating reactivities of cysteines in hsTop2␣ and probe the protein dynamics
Summary
The catalytic cycle of Top has multiple steps of reactions with corresponding conformational changes. Results: We can monitor conformational dynamics of hsTop2␣ upon binding with cofactors and the inhibitor ICRF-193. Most of the measured reactivities are in accordance with the predicted ones based on a homology structural model generated from available crystal structures. 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. On the basis of 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 the DNA gate between two closed clamp enzyme conformations locked by either 5-adenylyl ,␥-imidodiphosphate or the anticancer drug ICRF-193
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