The Impact of the Human DNA Topoisomerase II C-Terminal Domain on Activity

Background: Type II DNA topoisomerases (topos) are essential enzymes needed for the resolution of topological problems that occur during DNA metabolic processes. Topos carry out an ATP-dependent strand passage reaction whereby one double helix is passed through a transient break in another. Humans have two topoII isoforms, alpha and beta, which while enzymatically similar are differentially expressed and regulated, and are thought to have different cellular roles. The C-terminal domain (CTD) of the enzyme has the most diversity, and has been implicated in regulation. We sought to investigate the impact of the CTD domain on activity. Methodology/Principle Findings: We have investigated the role of the human topoII C-terminal domain by creating constructs encoding C-terminally truncated recombinant topoII alpha and beta and topoII alpha+beta-tail and topoII beta+alpha-tail chimeric proteins. We then investigated function in vivo in a yeast system, and in vitro in activity assays. We find that the C-terminal domain of human topoII isoforms is needed for in vivo function of the enzyme, but not needed for cleavage activity. C-terminally truncated enzymes had similar strand passage activity to full length enzymes, but the presence of the opposite C-terminal domain had a large effect, with the topoII alpha-CTD increasing activity, and the topoII beta-CTD decreasing activity. Conclusions/Significance: In vivo complementation data show that the topoII alpha C-terminal domain is needed for growth, but the topoII beta isoform is able to support low levels of growth without a C-terminal domain. This may indicate that topoII beta has an additional localisation signal. In vitro data suggest that, while the lack of any C-terminal domain has little effect on activity, the presence of either the topoII alpha or beta C-terminal domain can affect strand passage activity. Data indicates that the topoII beta-CTD may be a negative regulator. This is the first report of in vitro data with chimeric human topoIIs.