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

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

Schematic showing the position of the C-terminal domain of type II topoisomerases.

<p>Above each bar are the residue numbers at the start and end of the primary sequence, plus the point equating to the start of the C-terminal domain (indicated by arrows), as determined by limited proteolysis where known, and by alignment with this point where this is not known. Also shown are active site tyrosines (Y), known nuclear localisation sequences (NLS-dark grey) and known nuclear export sequences (NES–light grey). NLS sequences have been identified in human topoIIα (1259–1296, 1454–1497 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001754#pone.0001754-Mirski2" target="_blank">[20]</a>), human topoIIβ (1294–1332, 1522–1548, 1538–1573 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001754#pone.0001754-Mirski2" target="_blank">[20]</a>), <i>S. cerevisiae</i> topoII (1227–1242 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001754#pone.0001754-Caron1" target="_blank">[38]</a>) and <i>S. pombe</i> (26–44, 1227–1242, 1322–1339, 1335–1357 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001754#pone.0001754-Shiozaki1" target="_blank">[39]</a>). NES sequences have been identified in human topoIIα (1017–1028, 1054–1066 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001754#pone.0001754-Mirski3" target="_blank">[24]</a>) and human topoIIβ (1034–1044 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001754#pone.0001754-Mirski3" target="_blank">[24]</a>).</p

Protein parameters for human topoII isoforms

<p>Shown are the residues of the protein, the theoretical pI and number of acidic (negatively charged, D,E) and basic (positively charged, R,K) amino acids. Shown in parentheses is the percentage of amino acids with each charge in each protein. Reproduced and modified from KL Gilroy, thesis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001754#pone.0001754-Marsh1" target="_blank">[42]</a>.</p

Schematic of C-terminally truncated topoIIα and topoIIβ constructs.

<p>Shown are the topoII sequence boundaries, the GAL1 promoter, the 2μ replication origin, the URA3 marker gene, and an ampicillin resistance gene. Restriction sites used in plasmid construction are indicated.</p

Construction of chimeric ‘tail-swap’ plasmids.

<p>A–construction of topoIIα+β tail. TopoIIα fragments 30–791 and 792–1244 and topoIIβ fragment 1263–1621 were generated using restriction digests as shown, then ligated to give the final construct shown. B–construction of topoIIβ+α tail. TopoIIβ fragments 46–899 and 900–1263 and topoIIα fragment 1244–1531 were generated with restriction digests as indicated, then ligated to give the final construct shown <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001754#pone.0001754-Meczes1" target="_blank">[32]</a>.</p

Activity of recombinant proteins.

<p>A: Decatenation activity of all proteins, each column is the mean of at least two independent experiments. Standard errors are shown, with significant difference from full length enzyme marker with ‘***’. B: Representative cleavage experiment with 4.3 kb linearised pBR322 DNA with all proteins in the presence of mitoxantrone. TopoIIβ proteins in this case carry the S165R mutation.</p