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

K. L. Gilroy; Chrysoula Leontiou; Kay Padget; Jeremy Lakey; Caroline Austin

doi:10.1093/nar/gkl057

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

K. L. Gilroy, Chrysoula Leontiou, Kay Padget, Jeremy Lakey, 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.

Original language	English
Pages (from-to)	1597-1607
Journal	Nucleic Acids Research
Volume	34
Issue number	5
DOIs	https://doi.org/10.1093/nar/gkl057
Publication status	Published - 1 Jan 2006

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title = "mAMSA resistant human topoisomerase II mutation G465D has reduced ATP hydrolysis activity",

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.",

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T1 - mAMSA resistant human topoisomerase II mutation G465D has reduced ATP hydrolysis activity

AU - Gilroy, K. L.

AU - Leontiou, Chrysoula

AU - Padget, Kay

AU - Lakey, Jeremy

AU - Austin, Caroline

PY - 2006/1/1

Y1 - 2006/1/1

N2 - 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.

AB - 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.

UR - https://www.scopus.com/pages/publications/33645471331

U2 - 10.1093/nar/gkl057

DO - 10.1093/nar/gkl057

M3 - Article

SN - 0305-1048

VL - 34

SP - 1597

EP - 1607

JO - Nucleic Acids Research

JF - Nucleic Acids Research

IS - 5

ER -

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

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