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Mutation P732L in Human DNA Topoisomerase II Abolishes DNA Cleavage in the Presence of Calcium and Confers Drug Resistance

Institute for Cell and Molecular Biosciences, The Medical School, University of Newcastle upon Tyne, United Kingdom

The anti cancer drug methyl N-(4'-(9-acridinylamino)-3-methoxy-phenyl) methane sulfonamide (mAMSA) targets human DNA topoisomerase II. We report here the first selection with mAMSA of resistant human topoisomerase II. Random mutagenesis of human DNA topoisomerase II cDNA, followed by selection in yeast for resistance to mAMSA, identified P732L. This mutant was 10-fold less sensitive to mAMSA and cross-resistant to other chemotherapeutic agents such as etoposide, ellipticine, methyl N-(4'-(9-acridinylamino)-2-methoxy-phenyl) carbamate hydrochloride (mAMCA), methyl N-(4'-(9-acridinylamino)-phenyl) carbamate hydrochloride (AMCA), and doxorubicin. P732L is functional but has reduced strand passage activities and altered DNA binding compared with the wild-type protein. It has drastically altered cleavage properties compared with the wild-type enzyme. It cleaved a 40-base pair (bp) DNA substrate in the presence of magnesium but at positions different from that of the wild-type protein. More striking is that P732L was unable to cleave the 40-bp DNA substrate, a 500-bp linear substrate, or a 4.3-kilobase supercoiled substrate in the presence of calcium ions. This is the first report of a topoisomerase II mutation abolishing the ability of calcium to support DNA cleavage. This provides evidence for metal ion requirement for the phosphoryltransfer reaction of topoisomerase II and a possible mechanism for drug resistance.

Address correspondence to: Professor Caroline A. Austin, Lab 3011, Institute for Cell and Molecular Bioscience, The Medical School, University of Newcastle, Newcastle upon Tyne, NE2 4HH, UK. E-mail: caroline.austin{at}ncl.ac.uk

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