PT  - JOURNAL ARTICLE
AU  - Paola Fiorani
AU  - James F. Amatruda
AU  - Alessandra Silvestri
AU  - Richard H. Butler
AU  - Mary-Ann Bjornsti
AU  - Piero Benedetti
TI  - Domain Interactions Affecting Human DNA Topoisomerase I Catalysis and Camptothecin Sensitivity
AID  - 10.1124/mol.56.6.1105
DP  - 1999 Dec 01
TA  - Molecular Pharmacology
PG  - 1105--1115
VI  - 56
IP  - 6
4099  - http://molpharm.aspetjournals.org/content/56/6/1105.short
4100  - http://molpharm.aspetjournals.org/content/56/6/1105.full
SO  - Mol Pharmacol1999 Dec 01; 56
AB  - 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 humantop1 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.