A mutation in human topoisomerase II alpha whose expression is lethal in DNA repair-deficient yeast cells.

Published

Journal Article

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 alpha, 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 Top2alpha 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. hTop2alpha(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.

Full Text

Cited Authors

  • Walker, JV; Nitiss, KC; Jensen, LH; Mayne, C; Hu, T; Jensen, PB; Sehested, M; Hsieh, T; Nitiss, JL

Published Date

  • June 18, 2004

Published In

Volume / Issue

  • 279 / 25

Start / End Page

  • 25947 - 25954

PubMed ID

  • 15037624

Pubmed Central ID

  • 15037624

International Standard Serial Number (ISSN)

  • 0021-9258

Digital Object Identifier (DOI)

  • 10.1074/jbc.M312314200

Language

  • eng

Conference Location

  • United States