Characterisation of the DNA-dependent ATPase activity of human DNA topoisomerase IIβ: mutation of Ser165 in the ATPase domain reduces the ATPase activity and abolishes the in vivo complementation ability

We report for the first time an analysis of the ATPase activity of human DNA topoisomerase (topo) IIβ. We show that topo IIβ 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 IIβ in the presence of DNA is 2.25 s(–1). We have characterised a topo IIβ derivative which carries a mutation in the ATPase domain (S165R). S165R reduced the k(cat) 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 IIβ (k(cat)/K(mapp)) showed a 90% reduction for βS165R. 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 IIβ. Ser165 is predicted to lie very close to the bound nucleotide, and the S165R mutation could thus influence both ATP binding and ADP dissociation.