Nucleic Acids Research, 2002, Vol. 30, No. 24 5416-5424
© 2002 Oxford University Press
Characterisation of the DNA-dependent ATPase activity of human DNA topoisomerase IIß: mutation of Ser165 in the ATPase domain reduces the ATPase activity and abolishes the in vivo complementation ability
School of Cell and Molecular BioSciences, The Medical School, The University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, UK
*To whom correspondence should be addressed. Tel: +44 191 222 8864; Fax: +44 191 222 7424; Email: caroline.austin{at}ncl.ac.uk
Present address: Katherine L. West, Laboratory of Metabolism, Building 37, Room 2A19, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
We report for the first time an analysis of the ATPase activity of human DNA topoisomerase (topo) IIß. We show that topo IIß is a DNA-dependent ATPase that appears to fit Michaelis–Menten kinetics. The ATPase activity is stimulated 44-fold by DNA. The kcat for ATP hydrolysis by human DNA topo IIß in the presence of DNA is 2.25 s–1. We have characterised a topo IIß 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 Km. The specificity constant for the interaction between ATP and topo IIß (kcat/Kmapp) showed a 90% reduction for ßS165R. 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 IIß. Ser165 is predicted to lie very close to the bound nucleotide, and the S165R mutation could thus influence both ATP binding and ADP dissociation.