Domain interactions affecting human DNA topoisomerase I catalysis and camptothecin sensitivity

DNA topoisomerase I (Top1p) relaxes supercoiled DNA by the formation of a c ovalent intermediate in which the active site tyrosine is transiently bound to the severed DNA strand. The antineoplastic agent camptothecin (Cpt) spe cifically targets Top1p and several mutations have been isolated that rende r the enzyme Cpt resistant. The mutated residues, although located in diffe rent regions of the enzyme, may constitute part of the Cpt binding site. To begin identifying the structural features of DNA Top1p important for Cpt-i nduced cytotoxicity, we developed a novel yeast genetic screen to isolate c atalytically active, yet Cpt-resistant enzymes from a pool of human top1 mu tants. Among the mutations isolated were substitutions of Ser or Val for Gl y363, which like the Gly363 to Cys mutation previously reported by us, supp ressed the Cpt sensitivity of Top1p. In contrast, each amino-acid substitut ion 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 sup port a model where interactions between two conserved domains, a central "l ip" region containing residue Gly363 and the residues around the active sit e tyrosine (Tyr723), directly affect the formation of the Cpt-binding site and enzyme catalysis.