title: Characterisation of the DNA-dependent ATPase activity of human DNA topoisomerase IIbeta: mutation of Ser165 in the ATPase domain reduces the ATPase activity and abolishes the in vivo complementation ability.
abstract: We report for the first time an analysis of the ATPase activity of human DNA topoisomerase (topo) IIbeta. We show that topo IIbeta is a DNA-dependent ATPase that appears to fit Michaelis-Menten kinetics. The ATPase activity is stimulated 44-fold by DNA. The k(cat) for ATP hydrolysis by human DNA topo IIbeta in the presence of DNA is 2.25 s(-1). We have characterised a topo IIbeta derivative which carries a mutation in the ATPase domain (S165R). S165R reduced the kcat for ATP hydrolysis by 7-fold, to 0.32 s(-1), while not significantly altering the apparent K(m). The specificity constant for the interaction between ATP and topo IIbeta (kcat/K(mapp)) showed a 90% reduction for betaS165R. The DNA binding affinity and ATP-independent DNA cleavage activity of the enzyme are unaffected by this mutation. However, the strand passage activity is reduced by 80%, presumably due to reduced ATP hydrolysis. The mutant enzyme is unable to complement ts yeast topo II in vivo. We have used computer modelling to predict the arrangement of key residues at the ATPase active site of topo IIbeta. Ser165 is predicted to lie very close to the bound nucleotide, and the S165R mutation could thus influence both ATP binding and ADP dissociation.
authors.affiliation: School of Cell and Molecular BioSciences, The Medical School, The University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, UK.
authors.forename: Katherine L
authors.initials: KL
authors.lastname: West
authors.forename: Rosalind M
authors.initials: RM
authors.lastname: Turnbull
authors.forename: Elaine
authors.initials: E
authors.lastname: Willmore
authors.forename: Jeremy H
authors.initials: JH
authors.lastname: Lakey
authors.forename: Caroline A
authors.initials: CA
authors.lastname: Austin
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000494:pharmacology; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004305:Dose-Response Relationship, Drug; D005816:Genetic Complementation Test; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; D012694:Serine; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D004250:DNA Topoisomerases, Type II