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mAMSA resistant human topoisomerase IIβ mutation G465D has reduced ATP hydrolysis activity

Lookup NU author(s): Kathryn Gilroy, Chrysoula Leontiou, Dr Kay Padget, Professor Jeremy Lakey, Professor Caroline Austin

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Abstract

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. © 2006 Oxford University Press.


Publication metadata

Author(s): Gilroy KL, Leontiou C, Padget K, Lakey JH, Austin CA

Publication type: Article

Publication status: Published

Journal: Nucleic Acids Research

Year: 2006

Volume: 34

Issue: 5

Pages: 1597-1607

Print publication date: 01/01/2006

ISSN (print): 0305-1048

ISSN (electronic): 1362-4962

Publisher: Oxford University Press

URL: http://dx.doi.org/10.1093/nar/gkl057

DOI: 10.1093/nar/gkl057

PubMed id: 16549872


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