TY - JOUR
T1 - Domain interactions affecting human DNA topoisomerase I catalysis and camptothecin sensitivity
AU - Fiorani, Paola
AU - Amatruda, James F.
AU - Silvestri, Alessandra
AU - Butler, Richard H.
AU - Bjornsti, Mary Ann
AU - Benedetti, Piero
PY - 1999
Y1 - 1999
N2 - 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.
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 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.
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U2 - 10.1124/mol.56.6.1105
DO - 10.1124/mol.56.6.1105
M3 - Article
C2 - 10570037
AN - SCOPUS:0032752806
SN - 0026-895X
VL - 56
SP - 1105
EP - 1115
JO - Molecular Pharmacology
JF - Molecular Pharmacology
IS - 6
ER -