IngentaConnect Modulation of drug sensitivity in yeast cells by the ATP-binding ...

Authors: Nathalie Vilain; Monika Tsai-Pflugfelder¹; Audrey Benoit²; Susan M. Gasser¹; Didier Leroy³

Source: Nucleic Acids Research, Volume 31, Number 19, 01 October 2003 , pp. 5714-5722(9)

Abstract:

Epipodophyllotoxins are effective antitumour drugs that trap eukaryotic DNA topoisomerase II in a covalent complex with DNA. Based on DNA cleavage assays, the mode of interaction of these drugs was proposed to involve amino acid residues of the catalytic site. An in vitro binding study, however, revealed two potential binding sites for etoposide within human DNA topoisomerase II alpha

(htopoII

), one in the catalytic core of the enzyme and one in the ATP-binding N-terminal domain. Here we have tested how N-terminal mutations that reduce the affinity of the site for etoposide or ATP affect the sensitivity of yeast cells to etoposide. Surprisingly, when introduced into full-length enzymes, mutations that lower the drug binding capacity of the N-terminal domain in vitro render yeast more sensitive to epipodophyllotoxins. Consistently, when the htopoII alpha

N-terminal domain alone is overexpressed in the presence of yeast topoII, cells become more resistant to etoposide. Point mutations that weaken etoposide binding eliminate this resistance phenotype. We argue that the N-terminal ATP-binding pocket competes with the active site of the holoenzyme for binding etoposide both in cis and in trans with different outcomes, suggesting that each topoisomerase II monomer has two non-equivalent drug-binding sites.

Document Type: Research article

DOI: 10.1093/nar/gkg737

Affiliations: 1: Department of Molecular Biology and 2: NCCR Frontiers in Genetics, University of Geneva, Quai Ernest-Ansermet 30, CH-1211 Geneva, Switzerland and 3: *To whom correspondence should be addressed at NCCR Frontiers in Genetics, University of Geneva, Quai Ernest-Ansermet 30, CH-1211 Geneva, Switzerland. didier.leroy@frontiers-in-genetics.org, Tel: +41 22 379 65 90, Fax: +41 22 379 68 68

Modulation of drug sensitivity in yeast cells by the ATP-binding domain of human DNA topoisomerase II