title: A single mutation in the 729 residue modulates human DNA topoisomerase IB DNA binding and drug resistance.
abstract: Human DNA topoisomerase I (hTop1p) catalyzes the relaxation of supercoiled DNA and constitutes the cellular target of the antitumor drug camptothecin (CPT). The X-ray crystal structure of the enzyme covalently joined to DNA and bound to the CPT analog Topotecan suggests that there are two classes of mutations that can produce a CPT-resistant enzyme. The first class includes changes in residues that directly interact with the drug, whereas a second class alters interactions with the DNA and thereby destabilizes the drug binding site. The Thr729Ala, that is part of a hydrophobic pocket in the enzyme C-terminal domain, belongs to a third group of mutations that confer CPT resistance, but do not interact directly with the drug or the DNA. To understand the contribution of this residue in drug resistance, we have studied the effect on hTop1p catalysis and CPT sensitivity of four different substitutions in the Thr729 position (Thr729Ala, Thr729Glu, Thr729Lys and Thr729Pro). Tht729Glu and Thr729Lys mutants show severe CPT resistance and furthermore, Thr729Glu shows a remarkable defect in DNA binding. We postulate that the maintenance of the hydrophobic pocket integrity, where Thr729 is positioned, is crucial for drug sensitivity and DNA binding.
authors.affiliation: Department of Biology, University of Padova, Via U. Bassi 58/B, Padua 35131, Italy.
authors.forename: Carmen
authors.initials: C
authors.lastname: Losasso
authors.forename: Erica
authors.initials: E
authors.lastname: Cretaio
authors.forename: Paola
authors.initials: P
authors.lastname: Fiorani
authors.forename: Ilda
authors.initials: I
authors.lastname: D'Annessa
authors.forename: Giovanni
authors.initials: G
authors.lastname: Chillemi
authors.forename: Piero
authors.initials: P
authors.lastname: Benedetti
mesh_terms: D019943:Amino Acid Substitution; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D002166:Camptothecin / Q000494:pharmacology*; D002384:Catalysis; D004247:DNA / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism; D019008:Drug Resistance, Neoplasm; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D009154:Mutation; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013912:Threonine / Q000235:genetics; D059004:Topoisomerase I Inhibitors
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D004791:Enzyme Inhibitors; D059004:Topoisomerase I Inhibitors; D013912:Threonine; D004247:DNA; D004264:DNA Topoisomerases, Type I; C528909:TOP1 protein, human; D002166:Camptothecin
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