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

Abstract

BackgroundType 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, α and β, 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 FindingsWe have investigated the role of the human topoII C-terminal domain by creating constructs encoding C-terminally truncated recombinant topoIIα and β and topoIIα+β-tail and topoIIβ+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-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 topoIIα-CTD increasing activity, and the topoIIβ-CTD decreasing activity.Conclusions/Significance In vivo complementation data show that the topoIIα C-terminal domain is needed for growth, but the topoIIβ isoform is able to support low levels of growth without a C-terminal domain. This may indicate that topoIIβ 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 topoIIα or β C-terminal domain can affect strand passage activity. Data indicates that the topoIIβ-CTD may be a negative regulator. This is the first report of in vitro data with chimeric human topoIIs.

African swine fever virus ORF P1192R codes for a functional type II DNA topoisomerase

“…1). The importance of the C-terminal domain in enzymatic regulation has been previously reported, with sites and motifs present in this domain being responsible for regulating protein localization (Caron et al, 1994;Jensen et al, 1996;Mirski et al, 2003Mirski et al, , 1999, DNA-binding (Gilroy and Austin, 2011) and activity (Meczes et al, 2008). Still, the C-terminal domain is also absent from some previously described type II topoisomerases, as in the case of topoII from Chlorella viruses, and these have been shown to have high DNA cleavage activity (Dickey et al, 2005;Fortune et al, 2001).…”

Physiological Roles of DNA Double-Strand Breaks

“…Type II topoisomerase (Top II) activity in mammals is mediated by two isoenzymes, Top IIa and Top IIb, which exhibit considerable homology except in their less conserved C-termini [ 183 , 184 ] and display different subcellular distributions during the cell cycle [ 185 , 186 ]. Studies on Top IIa/b chimeric “tail swap” proteins and C-terminal truncations have shown that the C-terminal regions contribute to isoform-specific functionality and may modulate decatenation activity [ 187 , 188 ]. Immunohistochemical studies of Top II isoform expression patterns in normal and neoplastic human tissues show that Top IIa expression is largely restricted to normal proliferative tissue and is highly expressed in certain tumour types.…”

SUMO Modification of DNA topoisomerase II: Trying to get a CENse of it all

“…(B) Alignment of CTD sequences from human, mouse, Drosophila and yeast topo IIs. In these organisms, the boundary between the CTD and the cleavage/ligation domain has been mapped through limited proteolytic cleavage [23,180–183]. With the exception of human topo IIb (which starts right at the CTD boundary), this alignment is based on sequences starting 10 amino acids prior to the protease sensitive site.…”