Probing conformational changes in human DNA topoisomerase IIα by pulsed alkylation mass spectrometry.

Published

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Chen, Y-T; Collins, TRL; Guan, Z; Chen, VB; Hsieh, T-S

Published Date

  • July 20, 2012

Published In

Volume / Issue

  • 287 / 30

Start / End Page

  • 25660 - 25668

PubMed ID

  • 22679013

Pubmed Central ID

  • 22679013

Electronic International Standard Serial Number (EISSN)

  • 1083-351X

Digital Object Identifier (DOI)

  • 10.1074/jbc.M112.377606

Language

  • eng

Conference Location

  • United States