EXPRESSION, DOMAIN-STRUCTURE, AND ENZYMATIC-PROPERTIES OF AN ACTIVE RECOMBINANT HUMAN DNA TOPOISOMERASE II-BETA

被引:106
作者
AUSTIN, CA
MARSH, KL
WASSERMAN, RA
WILLMORE, E
SAYER, PJ
WANG, JC
FISHER, LM
机构
[1] HARVARD UNIV,DEPT MOLEC & CELLULAR BIOL,CAMBRIDGE,MA 02138
[2] UNIV LONDON ST GEORGES HOSP,SCH MED,DEPT CELLULAR & MOLEC SCI,MOLEC GENET GRP,LONDON SW17 0RE,ENGLAND
关键词
D O I
10.1074/jbc.270.26.15739
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Human cells express two genetically distinct isoforms of DNA topoisomerase II, alpha and beta, which catalyze ATP-dependent DNA strand passage and are an important antitumor drug target, Here we report for the first time the successful overexpression of human topoisomerase II beta in yeast by cloning a topoisomerase II beta cDNA in a yeast shuttle vector under the control of a galactose inducible promoter, Recombinant human topoisomerase II beta (residues 46-1621 fused to the first 5 residues of yeast topoisomerase II) was purified to homogeneity, yielding an enzymatically active polypeptide in sufficient quantity to allow analysis of its domain structure and comparison with that of recombinant human topoisomerase II alpha. Partial digestion of beta with either trypsin or protease SV8 generated fragments of approximately 130, 90, 62, and 45-50 kDa, arising from cleavage at three limited and discrete regions of the protein (A, B, and C) indicating the presence of at least four structural domains, Recombinant human topoisomerase II alpha and beta induced DNA breakage which was promoted by a variety of agents, Isoform differences in drug-induced DNA breakage were observed, These studies of human topoisomerase II beta in concert with alpha should aid the determination of their individual roles in cancer chemotherapy and should facilitate the design, targeting, and testing of cytotoxic antitumor agents.
引用
收藏
页码:15739 / 15746
页数:8
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