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

Meczes EL, Gilroy KL, West KL, Austin CA - PLoS ONE (2008)

Bottom Line: 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 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.This is the first report of in vitro data with chimeric human topoIIs.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cell and Molecular Biosciences, The University of Newcastle upon Tyne, Newcastle Upon Tyne, United Kingdom.

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 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 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.

Conclusions/significance: 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.

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Activity of recombinant proteins.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.
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pone-0001754-g003: Activity of recombinant proteins.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.

Mentions: The decatenation activity of wild type topoIIα, C-terminally truncated topoIIα, and topoIIα+β tail was assayed. The values for 50% decatenation (D50), in ng of protein, are shown in figure 3A. There is no significant difference in D50 between topoIIα and its C-terminal truncation, with values of 4.5±1.3 and 6±1 ng of protein respectively. The topoIIα+β tail chimera however does show a significant reduction in decatenation as compared to full length topoIIα, with a D50 of 29±1 (p = 0.0008 in a two-tailed unpaired student t-test). This implies that while topoIIα can function perfectly well without a C-terminal domain, the β-tail on the topoIIα enzyme impedes activity.


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

Meczes EL, Gilroy KL, West KL, Austin CA - PLoS ONE (2008)

Activity of recombinant proteins.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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2262138&req=5

pone-0001754-g003: Activity of recombinant proteins.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.
Mentions: The decatenation activity of wild type topoIIα, C-terminally truncated topoIIα, and topoIIα+β tail was assayed. The values for 50% decatenation (D50), in ng of protein, are shown in figure 3A. There is no significant difference in D50 between topoIIα and its C-terminal truncation, with values of 4.5±1.3 and 6±1 ng of protein respectively. The topoIIα+β tail chimera however does show a significant reduction in decatenation as compared to full length topoIIα, with a D50 of 29±1 (p = 0.0008 in a two-tailed unpaired student t-test). This implies that while topoIIα can function perfectly well without a C-terminal domain, the β-tail on the topoIIα enzyme impedes activity.

Bottom Line: 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 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.This is the first report of in vitro data with chimeric human topoIIs.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cell and Molecular Biosciences, The University of Newcastle upon Tyne, Newcastle Upon Tyne, United Kingdom.

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 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 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.

Conclusions/significance: 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.

Show MeSH