DNA Topoisomerase I Domain Interactions Impact Enzyme Activity and Sensitivity to Camptothecin

• Published in: The Journal of biological chemistry Vol. 290; no. 19; pp. 12068 - 12078
• Main Authors: Wright, Christine M., van der Merwe, Marié, DeBrot, Amanda H., Bjornsti, Mary-Ann
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 08.05.2015; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Anticancer Drug; Camptothecin; Camptothecin - chemistry; Catalysis; DNA Topoisomerase; DNA Topoisomerases, Type I - chemistry; Enzymology; Humans; Models, Molecular; Molecular Sequence Data; Protein Chimera; Protein Domain; Protein Structure, Tertiary; Recombinant Fusion Proteins - chemistry; Saccharomyces cerevisiae; Saccharomyces cerevisiae - enzymology; Sequence Homology, Amino Acid; Species Specificity; Topoisomerase I Inhibitors - chemistry; Protein Chimera; Camptothecin; Protein Domain; Saccharomyces cerevisiae; Anticancer Drug; DNA Topoisomerase
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.635078

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 Gly717 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. Background: Despite similarities in mechanism and architecture, human DNA topoisomerase I (Top1) is ∼100-fold more sensitive to camptothecin (CPT) than yeast Top1. Results: Reciprocal swaps of conserved and divergent protein domains alter chimeric Top1 activity. Conclusion: Conserved core and C-terminal domains dictate Top1 biochemical behavior and intrinsic CPT sensitivity. Significance: Interactions between nonconserved structural domains of Top1 impair cell viability, independent of enzyme catalysis.