AMSA resistant human topoisomerase IIbeta mutation G465D has reduced ATP hydrolysis activity. Nucleic Acids Res 34(5): 1597-1607

The Institute for Cell and Molecular Biosciences, The University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK.
Nucleic Acids Research (Impact Factor: 9.11). 02/2006; 34(5):1597-607. DOI: 10.1093/nar/gkl057
Source: PubMed


Type II Human DNA Topoisomerases (topos II) play an essential role in DNA replication and transcription and are important
targets for cancer chemotherapeutic drugs. Topoisomerase II causes transient double-strand breaks in DNA, forming a gate through
which another double helix is passed, and acts as a DNA dependent ATPase. Mutations in topoII have been linked to atypical
multi-drug resistance. Both human Topoisomerase II isoforms, α and β, are targeted by amsacrine. We have used a forced molecular
evolution approach to identify mutations conferring resistance to acridines. Here we report mutation βG465D, which was selected
with mAMSA and DACA and is cross-resistant to etoposide, ellipticine and doxorubicin. Resistance to mAMSA appears to decrease
over time indicating a previously unreported resistance mechanism. G465D lies within the B′ domain in the region that contacts
the cleaved gate helix. There is a 3-fold decrease in ATP affinity and ATP hydrolysis and an altered requirement for magnesium
in decatenation assays. The decatenation rate is decreased for the mutated G465D protein. And we report for the first time
the use of fluorescence anisotropy with intact human topoisomerase II.

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Available from: Kay Padget, Jan 26, 2016
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    • "Proteins were overexpressed and purified from S.cerevisiae strain JEL1Δtop1 as described previously [34]–[35]. Full length topoisomerase IIα was expressed from plasmid YEpWob6 [36] and full length topoisomerase IIβ was expressed from plasmid YEphTOP2βKLM [37]. Truncated topoisomerase IIα and truncated topoisomerase IIβ were expressed from plasmids YEphTOP2αt(1242) and YEphTOP2βt(1263) respectively, as we reported previously [33]. "
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    ABSTRACT: Type II DNA topoisomerases are essential, ubiquitous enzymes that act to relieve topological problems arising in DNA from normal cellular activity. Their mechanism of action involves the ATP-dependent transport of one DNA duplex through a transient break in a second DNA duplex; metal ions are essential for strand passage. Humans have two isoforms, topoisomerase IIα and topoisomerase IIβ, that have distinct roles in the cell. The C-terminal domain has been linked to isoform specific differences in activity and DNA interaction. We have investigated the role of the C-terminal domain in the binding of human topoisomerase IIα and topoisomerase IIβ to DNA in fluorescence anisotropy assays using full length and C-terminally truncated enzymes. We find that the C-terminal domain of topoisomerase IIβ but not topoisomerase IIα affects the binding of the enzyme to the DNA. The presence of metal ions has no effect on DNA binding. Additionally, we have examined strand passage of the full length and truncated enzymes in the presence of a number of supporting metal ions and find that there is no difference in relative decatenation between isoforms. We find that calcium and manganese, in addition to magnesium, can support strand passage by the human topoisomerase II enzymes. The C-terminal domain of topoisomerase IIβ, but not that of topoisomerase IIα, alters the enzyme's K(D) for DNA binding. This is consistent with previous data and may be related to the differential modes of action of the two isoforms in vivo. We also show strand passage with different supporting metal ions for human topoisomerase IIα or topoisomerase IIβ, either full length or C-terminally truncated. They all show the same preferences, whereby Mg > Ca > Mn.
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    • "Decatenation assays and cleavage assays with an end-labelled 4.3 kb linear DNA fragment from pBR322 were done as described previously [29]–[30]. "
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    ABSTRACT: 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.
    Preview · Article · Feb 2008 · PLoS ONE
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    ABSTRACT: Electrospun nanofibers could be used to mimic the nanofibrous structure of the extracellular matrix (ECM) in native tissue. In tissue engineering, the ECM could be used as tissue engineering scaffold to solve tissue engineering problems. In this paper, poly(L-lactid-co-ɛ-caprolactone) [P(LLA-CL)] nanofibers and collagen-chitosan complex nanofibers were fabricated by electrospinning. Results of the experiments showed that the mechanical properties of the collagen-chitosan complex nanofibers varied with the collagen content in the complex. It was also found that the biodegradability of P(LLA-CL) nanofibers was faster than its membrane and that smooth muscle cells (SMC) grow faster on collagen nanofibers than on P(LLA-CL) nanofibers.
    No preview · Article · Jan 2007 · Frontiers of Materials Science in China
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