title: The DNA dependence of the ATPase activity of human DNA topoisomerase IIalpha.
abstract: We have purified human topoisomerase IIalpha from HeLa cells and studied its ATPase reaction. The ATPase activity is stimulated by DNA and shows apparent Michaelis-Menten kinetics. Although the ATPase activity of human topoisomerase IIalpha is lower than that of Saccharomyces cerevisiae, it is more active in decatenation, implying more efficient coupling of the ATPase to DNA strand passage under these conditions. Using plasmid pBR322 as the DNA cofactor, the reaction shows hyperstimulation by DNA at a base pair to enzyme dimer ratio of 100-200:1. When DNA fragments are used as the cofactor, the reaction requires > approximately 100 base pairs to stimulate the activity and fragments of approximately 300 base pairs show hyperstimulation. This behavior can be rationalized in terms of the enzyme requiring fragments that can bind to both the DNA gate and the ATP-operated clamp in order for the ATPase reaction to be stimulated. Hyperstimulation is a consequence of the saturation of DNA with enzyme. The mechanistic implications of these results are discussed.
authors.affiliation: Department of Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom.
authors.forename: T R
authors.initials: TR
authors.lastname: Hammonds
authors.forename: A
authors.initials: A
authors.lastname: Maxwell
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000951:Antigens, Neoplasm; D004247:DNA / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000302:isolation & purification / Q000378:metabolism*; D004268:DNA-Binding Proteins; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics
chemical_list: D000951:Antigens, Neoplasm; D004268:DNA-Binding Proteins; D007527:Isoenzymes; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases; D004250:DNA Topoisomerases, Type II