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J. Biol. Chem., Vol. 279, Issue 25, 25947-25954, June 18, 2004

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A Mutation in Human Topoisomerase II Whose Expression Is Lethal in DNA Repair-deficient Yeast Cells^*

Jerrylaine V. Walker, Karin C. Nitiss, Lars H. Jensen, Christopher Mayne, Tao Hu¶, Peter B. Jensen||, Maxwell Sehested, Tao Hsieh¶, and John L. Nitiss**

From the Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, the Department of Pathology, Laboratory Center, Rigshospitalet 5444, DK-2100 Copenhagen, Denmark, the ^¶Department of Biochemistry, Duke University, Durham, North Carolina 27710, and ^||Laboratory for Experimental Medical Oncology, Finsen Center, Rigshospitalet 5074, DK-2100 Copenhagen, Denmark

Type II DNA topoisomerases are ATP-dependent enzymes that catalyze alterations in DNA topology. These enzymes are important targets of a variety of anti-bacterial and anti-cancer agents. We identified a mutation in human topoisomerase II , changing aspartic acid 48 to asparagine, that has the unique property of failing to transform yeast cells deficient in recombinational repair. In repair-proficient yeast strains, the Asp-48 Asn mutant can be expressed and complements a temperature-sensitive top2 mutation. Purified Asp-48 Asn Top2 has relaxation and decatenation activity similar to the wild type enzyme, but the purified protein exhibits several biochemical alterations compared with the wild type enzyme. The mutant enzyme binds both covalently closed and linear DNA with greater avidity than the wild type enzyme. hTop2(Asp-48 Asn) also exhibited elevated levels of drug-independent cleavage compared with the wild type enzyme. The enzyme did not show altered sensitivity to bisdioxopiperazines nor did it form stable closed clamps in the absence of ATP, although the enzyme did form elevated levels of closed clamps in the presence of a non-hydrolyzable ATP analog compared with the wild type enzyme. We suggest that the lethality exhibited by the mutant is likely because of its enhanced drug-independent cleavage, and we propose that alterations in the ATP binding domain of the enzyme are capable of altering the interactions of the enzyme with DNA. This mutant enzyme also serves as a new model for understanding the action of drugs targeting topoisomerase II.

Received for publication, November 10, 2003 , and in revised form, March 1, 2004.

^* This work was supported by Grants CA82313 and CA52814 (to J. L. N.) from the National Institutes of Health, Core Grant CA21765, and the American Lebanese Syrian Associated Charities. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed: Dept. of Molecular Pharmacology, St. Jude Children's Research Hospital, 332 N. Lauderdale St., Memphis, TN 38105-2794. Tel.: 901-495-2794; Fax: 901-495-4290; E-mail: John.nitiss{at}stjude.org.

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