Using a biochemical approach to identify the primary dimerization regions in human DNA topoisomerase IIalpha.
Eukaryotic topoisomerase II is a nuclear enzyme essential for DNA metabolism and chromosome dynamics. The enzyme has a dimeric structure, and subunit dimerization is vital to the cellular functions and activities of the enzyme. Two biochemical approaches based on metal ion affinity chromatography and immunoprecipitation have been carried out to map the dimerization region(s) in human topoisomerase IIalpha. The results demonstrate that two regions spanning amino acids 1053-1069 and 1124-1143 are both essential for dimerization. The regions correspond to the interaction domains revealed in yeast topoisomerase II after crystallization of a central fragment of this enzyme, indicating that the overall C-terminal dimerization structure of eukaryotic topoisomerase II is conserved from yeast to human. Furthermore, linker insertion analysis has demonstrated that the two dimerization regions are located in a highly flexible part of the enzyme. Topoisomerase IIalpha mutant enzymes unable to dimerize via the C-terminal primary dimerization regions due to lack of one of the defined dimerization regions can still be forced to dimerize if DNA and an ATP analog are added to the reaction mixture. The result indicates that secondary interactions occur by ATP analog-mediated clamp closing when the subunits are brought together on DNA.
Bjergbaek
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Department of Molecular and Structural Biology, University of Aarhus, C. F. Mollers Allé, Building 130, 8000 Arhus C, Denmark.
Jensen
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Westergaard
O
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Andersen
A H
AH
D000595:Amino Acid Sequence; D000951:Antigens, Neoplasm; D001483:Base Sequence; D002846:Chromatography, Affinity; D017931:DNA Primers; D004250:DNA Topoisomerases, Type II / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins; D019281:Dimerization; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011233:Precipitin Tests
D000951:Antigens, Neoplasm; D017931:DNA Primers; D004268:DNA-Binding Proteins; D007527:Isoenzymes; D004250:DNA Topoisomerases, Type II