The Impact of the C-Terminal Domain on the Interaction of Human DNA Topoisomerase II [alpha] and [beta] with DNA

Type II DNA topoisomerases are essential, ubiquitous enzymes that act to relieve topological problems arising in DNA from normal cellular activity. Their mechanism of action involves the ATP-dependent transport of one DNA duplex through a transient break in a second DNA duplex; metal ions are essential for strand passage. Humans have two isoforms, topoisomerase II[alpha] and topoisomerase II[beta], that have distinct roles in the cell. The C-terminal domain has been linked to isoform specific differences in activity and DNA interaction. We have investigated the role of the C-terminal domain in the binding of human topoisomerase II[alpha] and topoisomerase II[beta] to DNA in fluorescence anisotropy assays using full length and C-terminally truncated enzymes. We find that the C-terminal domain of topoisomerase II[beta] but not topoisomerase II[alpha] affects the binding of the enzyme to the DNA. The presence of metal ions has no effect on DNA binding. Additionally, we have examined strand passage of the full length and truncated enzymes in the presence of a number of supporting metal ions and find that there is no difference in relative decatenation between isoforms. We find that calcium and manganese, in addition to magnesium, can support strand passage by the human topoisomerase II enzymes. The C-terminal domain of topoisomerase II[beta], but not that of topoisomerase II[alpha], alters the enzyme's K.sub.D for DNA binding. This is consistent with previous data and may be related to the differential modes of action of the two isoforms in vivo. We also show strand passage with different supporting metal ions for human topoisomerase II[alpha] or topoisomerase II[beta], either full length or C-terminally truncated. They all show the same preferences, whereby Mg > Ca > Mn.