Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Biol Chem. 2012 Jul 20;287(30):25660-8. doi: 10.1074/jbc.M112.377606. Epub 2012 Jun 7.
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.
II 型拓扑异构酶是通过将一段 DNA 双链穿过第二段的瞬时双链断裂来解决 DNA 拓扑问题的必需酶。该反应要求酶精确控制 DNA 切割和门控打开，同时伴随着 ATP 水解。我们使用脉冲烷基化质谱法，通过测量与单溴代丁二酰亚胺的巯基反应性，能够监测分布在人拓扑异构酶 IIα 中的 13 个半胱氨酸周围的溶剂可及性。大多数测量的反应性与基于可用晶体结构生成的同源结构模型预测的反应性一致。然而，这些结果为结构模型未涵盖的残基以及模型化和溶液全酶结构之间的潜在差异提供了新的信息。此外，基于位于 N 门和 DNA 门的几个半胱氨酸的反应性变化，我们可以在辅因子存在的情况下监测拓扑异构酶 II 的运动，并检测两种封闭夹酶构象之间的 DNA 门的差异，这两种构象分别由 5'-腺苷酰基 β，γ-亚胺二磷酸或抗癌药物 ICRF-193 锁定。
