Probing conformational changes in human DNA topoisomerase IIα by pulsed alkylation mass spectrometry.
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.
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Yu-Tsung
YT
Chen
Tammy R L
TR
Collins
Ziqiang
Z
Guan
Vincent B
VB
Chen
Tao-Shih
TS
Hsieh
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000478:Alkylation; D000951:Antigens, Neoplasm / Q000737:chemistry* / Q000378:metabolism; D001643:Bridged Bicyclo Compounds / Q000737:chemistry; D053903:DNA Breaks, Double-Stranded; D004250:DNA Topoisomerases, Type II / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D054659:Diketopiperazines; D020035:Holoenzymes / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D013058:Mass Spectrometry; D008958:Models, Molecular; D010879:Piperazines / Q000737:chemistry; D017434:Protein Structure, Tertiary
D000951:Antigens, Neoplasm; D001643:Bridged Bicyclo Compounds; D004268:DNA-Binding Proteins; D054659:Diketopiperazines; D020035:Holoenzymes; D010879:Piperazines; C070899:4,4'-(1,2-dimethyl-1,2-ethanediyl)bis-2,6-piperazinedione; D000255:Adenosine Triphosphate; D004250:DNA Topoisomerases, Type II; C028381:monobromobimane