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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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Schematic of C-terminally truncated topoIIα and topoIIβ constructs.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.
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pone-0001754-g001: Schematic of C-terminally truncated topoIIα and topoIIβ constructs.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.

Mentions: Truncations at the 3′ end of the coding sequence of topoIIα and topoIIβ (S165R) were constructed from plasmids YEpWob6 and YEphTOP2β respectively. The truncated topoIIα plasmid encodes residues 29–1242 and the truncated topoIIβ plasmid encodes residues 46–1263, these being the start of the C-terminal domains as determined by limited proteolysis experiments [28], [32], [33]. Schematics of these plasmids are shown in Figure 1.


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

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

Schematic of C-terminally truncated topoIIα and topoIIβ constructs.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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001754-g001: Schematic of C-terminally truncated topoIIα and topoIIβ constructs.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.
Mentions: Truncations at the 3′ end of the coding sequence of topoIIα and topoIIβ (S165R) were constructed from plasmids YEpWob6 and YEphTOP2β respectively. The truncated topoIIα plasmid encodes residues 29–1242 and the truncated topoIIβ plasmid encodes residues 46–1263, these being the start of the C-terminal domains as determined by limited proteolysis experiments [28], [32], [33]. Schematics of these plasmids are shown in Figure 1.

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
Related in: MedlinePlus