Domain interactions affecting human DNA topoisomerase I catalysis and camptothecin sensitivity

Paola Fiorani, James F. Amatruda, Alessandra Silvestri, Richard H. Butler, Mary Ann Bjornsti, Piero Benedetti

Research output: Contribution to journalArticle

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Abstract

DNA topoisomerase I (Top1p) relaxes supercoiled DNA by the formation of a covalent intermediate in which the active site tyrosine is transiently bound to the severed DNA strand. The antineoplastic agent camptothecin (Cpt) specifically targets Top1p and several mutations have been isolated that render the enzyme Cpt resistant. The mutated residues, although located in different regions of the enzyme, may constitute part of the Cpt binding site. To begin identifying the structural features of DNA Top1p important for Cpt- induced cytotoxicity, we developed a novel yeast genetic screen to isolate catalytically active, yet Cpt-resistant enzymes from a pool of human top1 mutants. Among the mutations isolated were substitutions of Ser or Val for Gly363, which like the Gly363 to Cys mutation previously reported by us, suppressed the Cpt sensitivity of Top1p. In contrast, each amino-acid substitution differed in its ability to suppress the lethal phenotype and catalytic activity of a human top1 mutant top1T718A that resembles Cpt by stabilizing the covalent intermediate. Biochemical analyses and molecular modeling support a model where interactions between two conserved domains, a central 'lip' region containing residue Gly363 and the residues around the active site tyrosine (Tyr723), directly affect the formation of the Cpt- binding site and enzyme catalysis.

Original languageEnglish (US)
Pages (from-to)1105-1115
Number of pages11
JournalMolecular Pharmacology
Volume56
Issue number6
StatePublished - 1999

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Camptothecin
Catalysis
Enzymes
Mutation
Tyrosine
Catalytic Domain
Binding Sites
Superhelical DNA
Type I DNA Topoisomerase
human TOP1 protein
DNA
Amino Acid Substitution
Lip
Human Activities
Antineoplastic Agents
Yeasts
Phenotype

ASJC Scopus subject areas

  • Pharmacology

Cite this

Fiorani, P., Amatruda, J. F., Silvestri, A., Butler, R. H., Bjornsti, M. A., & Benedetti, P. (1999). Domain interactions affecting human DNA topoisomerase I catalysis and camptothecin sensitivity. Molecular Pharmacology, 56(6), 1105-1115.

Domain interactions affecting human DNA topoisomerase I catalysis and camptothecin sensitivity. / Fiorani, Paola; Amatruda, James F.; Silvestri, Alessandra; Butler, Richard H.; Bjornsti, Mary Ann; Benedetti, Piero.

In: Molecular Pharmacology, Vol. 56, No. 6, 1999, p. 1105-1115.

Research output: Contribution to journalArticle

Fiorani, P, Amatruda, JF, Silvestri, A, Butler, RH, Bjornsti, MA & Benedetti, P 1999, 'Domain interactions affecting human DNA topoisomerase I catalysis and camptothecin sensitivity', Molecular Pharmacology, vol. 56, no. 6, pp. 1105-1115.
Fiorani P, Amatruda JF, Silvestri A, Butler RH, Bjornsti MA, Benedetti P. Domain interactions affecting human DNA topoisomerase I catalysis and camptothecin sensitivity. Molecular Pharmacology. 1999;56(6):1105-1115.
Fiorani, Paola ; Amatruda, James F. ; Silvestri, Alessandra ; Butler, Richard H. ; Bjornsti, Mary Ann ; Benedetti, Piero. / Domain interactions affecting human DNA topoisomerase I catalysis and camptothecin sensitivity. In: Molecular Pharmacology. 1999 ; Vol. 56, No. 6. pp. 1105-1115.
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