title: DNA topoisomerase I domain interactions impact enzyme activity and sensitivity to camptothecin.
abstract: During processes such as DNA replication and transcription, DNA topoisomerase I (Top1) catalyzes the relaxation of DNA supercoils. The nuclear enzyme is also the cellular target of camptothecin (CPT) chemotherapeutics. Top1 contains four domains: the highly conserved core and C-terminal domains involved in catalysis, a coiled-coil linker domain of variable length, and a poorly conserved N-terminal domain. Yeast and human Top1 share a common reaction mechanism and domain structure. However, the human Top1 is ∼100-fold more sensitive to CPT. Moreover, substitutions of a conserved Gly(717) residue, which alter intrinsic enzyme sensitivity to CPT, induce distinct phenotypes in yeast. To address the structural basis for these differences, reciprocal swaps of yeast and human Top1 domains were engineered in chimeric enzymes. Here we report that intrinsic Top1 sensitivity to CPT is dictated by the composition of the conserved core and C-terminal domains. However, independent of CPT, biochemically similar chimeric enzymes produced strikingly distinct phenotypes in yeast. Expression of a human Top1 chimera containing the yeast linker domain proved toxic, even in the context of a catalytically inactive Y723F enzyme. Lethality was suppressed either by splicing the yeast N-terminal domain into the chimera, deleting the human N-terminal residues, or in enzymes reconstituted by polypeptide complementation. These data demonstrate a functional interaction between the N-terminal and linker domains, which, when mispaired between yeast and human enzymes, induces cell lethality. Because toxicity was independent of enzyme catalysis, the inappropriate coordination of N-terminal and linker domains may induce aberrant Top1-protein interactions to impair cell growth.
authors: From the Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, Alabama 35294 and.
authors: Christine M
authors: CM
authors: Wright
authors: Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105.
authors: Marié
authors: M
authors: van der Merwe
authors: From the Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, Alabama 35294 and.
authors: Amanda H
authors: AH
authors: DeBrot
authors: From the Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, Alabama 35294 and bjornsti@uab.edu.
authors: Mary-Ann
authors: MA
authors: Bjornsti
mesh_terms: D000595:Amino Acid Sequence; D002166:Camptothecin / Q000737:chemistry*; D002384:Catalysis; D004264:DNA Topoisomerases, Type I / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D059004:Topoisomerase I Inhibitors / Q000737:chemistry*
chemical_list: D011993:Recombinant Fusion Proteins; D059004:Topoisomerase I Inhibitors; D004264:DNA Topoisomerases, Type I; C528909:TOP1 protein, human; D002166:Camptothecin
keywords: Anticancer Drug; Camptothecin; DNA Topoisomerase; Protein Chimera; Protein Domain; Saccharomyces cerevisiae