The impact of the human DNA topoisomerse II C-terminal domain on activity

Abstract

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 topoll isoforms, α and β, which while enzymatically similar are differentially expressed and regulated, and are thought to have different cellular roles. The C-role 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 topoll C-terminal by creating constructs encoding C-terminally truncated recombinant topollα and β and topollα+β-tail and topoll±α-tail chimeric proteins. We then investigated function in vivo in a yeast system, and in vitro in activity assays. We find that the C-terminally truncated enzymes had similar strand passage activity to full length enzymes, but the presense of the opposite C-terminal domain had a large effect, with the topollα-CTD increasing activity, and the topollβ-CTD decreasing activity. Conclusion/Significance: In vivo complementation data show that the topollα C-terminal domain is needed for growth, but the topollβ isoform is able to support low levels of growth without a C-terminal domain. This may indicate that topollβ 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 topollα or β C-terminal domain can affect strand passage activity. Data indicates that the topollβ-CTD may be a negative regulator. This is the first report in vitro data with chimeric human topolls. © 2008 Meczes et al.