mAMSA resistant human topoisomerase IIbeta mutation G465D has reduced ATP hydrolysis activity.
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, alpha and beta, are targeted by amsacrine. We have used a forced molecular evolution approach to identify mutations conferring resistance to acridines. Here we report mutation betaG465D, 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.
The Institute for Cell and Molecular Biosciences, The University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK.
Kathryn L
KL
Gilroy
Chrysoula
C
Leontiou
Kay
K
Padget
Jeremy H
JH
Lakey
Caroline A
CA
Austin
D000166:Acridines / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000677:Amsacrine / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000235:genetics* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D019020:Directed Molecular Evolution; D004351:Drug Resistance / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005454:Fluorescence Polarization; D006801:Humans; D006868:Hydrolysis; D015636:Magnesium Chloride / Q000494:pharmacology; D017354:Point Mutation
D000166:Acridines; D000970:Antineoplastic Agents; C121181:DACA, acridine; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D000677:Amsacrine; D015636:Magnesium Chloride; D000255:Adenosine Triphosphate; D004247:DNA; D004250:DNA Topoisomerases, Type II