Single Mutation in the Linker Domain Confers Protein Flexibility and Camptothecin Resistance to Human Topoisomerase I

Fiorani, Paola; Bruselles, Alessandro; Falconi, Mattia; Chillemi, Giovanni; Desideri, Alessandro; Benedetti, Piero

doi:10.1074/jbc.m303899200

Cited by 84 publications

(99 citation statements)

References 32 publications

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“…Moreover, specific topoisomerase I activity of the native resistant cell lines was fairly lower than that of sensitive cells, suggesting that immunoreactive topoisomerase I protein contains low levels of active form enzyme (54). Recently, gene sequencing revealed the presence of several mutations, potentially associated with drug resistance, and mainly involving exons 12, 13, 15, and 20 (55). Therefore, although similar data are still lacking for pancreatic cancer, it is conceivable that mutational analysis of topoisomerase I might be considered to exclude patients from receiving irinotecan if their genotype suggests drug resistance.…”

Section: Pharmacogenetics Of Pancreatic Cancermentioning

confidence: 99%

Pharmacogenetics of anticancer drug sensitivity in pancreatic cancer

Giovannetti

Mey

Nannizzi

et al. 2006

Molecular Cancer Therapeutics

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Chemotherapy has produced unsatisfactory results in pancreas cancer and novel approaches, including treatment tailoring by pharmacogenetic analysis and new molecular-targeted drugs, are required. The scarcity of effective therapies may reflect the lack of knowledge about the influence of tumor-related molecular abnormalities on responsiveness to drugs. Advances in the understanding of pancreas cancer biology have been made over the past decade, including the discovery of critical mutations in oncogenes (i.e., K-Ras) as well as the loss of tumor suppressor genes, such as TP53 and p16 INK4 . Other studies showed the dysregulation of the expression of proteins involved in the control of cell cycle, proliferation, apoptosis, and invasiveness, such as Bcl-2, Akt, mdm2, and epidermal growth factor receptor. These characteristics might contribute to the aggressive behavior of pancreatic cancer and influence response to treatment.

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Section: Pharmacogenetics Of Pancreatic Cancermentioning

confidence: 99%

Pharmacogenetics of anticancer drug sensitivity in pancreatic cancer

Giovannetti

Mey

Nannizzi

et al. 2006

Molecular Cancer Therapeutics

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“…For instance, when Topo70 is reconstituted from separate polypeptides comprising the Top1p core and the linker/C-terminal domains, the enzyme is catalytically active yet exhibits reduced sensitivity to CPT (26). Within the N-terminal ␣-helix of the linker, molecular dynamic simulations suggest mutation of Ala 653 to Pro (A 653 P) increases the flexibility of this coiled-coil structure, to enhance the rate of DNA religation and Top1p resistance to CPT (30). More direct evidence for physical interactions between the Top1p linker and active site come from recent studies where the combination of the A 653 P mutation with the self-poisoning T 722 A mutation suppressed the lethal phenotype of Top1T 722 A (32).…”

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confidence: 99%

Mutation of Gly721 Alters DNA Topoisomerase I Active Site Architecture and Sensitivity to Camptothecin

Merwe

Bjornsti

2008

Journal of Biological Chemistry

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DNA topoisomerase I (Top1p) catalyzes the relaxation of supercoiled DNA via a concerted mechanism of DNA strand cleavage and religation. Top1p is the cellular target of the anticancer drug camptothecin (CPT), which reversibly stabilizes a covalent enzyme-DNA intermediate. Top1p clamps around duplex DNA, wherein the core and C-terminal domains are connected by extended ␣-helices (linker domain), which position the active site Tyr of the C-terminal domain within the catalytic pocket. The physical connection of the linker with the Top1p clamp as well as linker flexibility affect enzyme sensitivity to CPT. Crystallographic data reveal that a conserved Gly residue (located at the juncture between the linker and C-terminal domains) is at one end of a short ␣-helix, which extends to the active site Tyr covalently linked to the DNA. In the presence of drug, the linker is rigid and this ␣-helix extends to include Gly and the preceding Leu. We report that mutation of this conserved Gly in yeast Top1p alters enzyme sensitivity to CPT. Mutating Gly to Asp, Glu, Asn, Gln, Leu, or Ala enhanced enzyme CPT sensitivity, with the acidic residues inducing the greatest increase in drug sensitivity in vivo and in vitro. By contrast, Val or Phe substituents rendered the enzyme CPTresistant. Mutation-induced alterations in enzyme architecture preceding the active site Tyr suggest these structural transitions modulate enzyme sensitivity to CPT, while enhancing the rate of DNA cleavage. We postulate that this conserved Gly residue provides a flexible hinge within the Top1p catalytic pocket to facilitate linker dynamics and the structural alterations that accompany drug binding of the covalent enzyme-DNA intermediate.Eukaryotic DNA topoisomerase 1 (Top1p) 2 alters DNA topology by introducing a transient break in a single strand of the DNA duplex, thereby allowing strand rotation at the site of DNA scission to relieve superhelical tension (1-3). The enzyme plays critical roles in processes such as replication, transcription, recombination, and chromosomal condensation, with a reaction mechanism highly conserved from yeast to human. The active site tyrosine (Tyr 727 in yeast) acts as a nucleophile to cleave a single DNA strand, forming a covalent phosphotyrosyl linkage between the enzyme and the 3Ј-end of the DNA. The free 5Ј-OH end of the scissile strand then rotates about the uncleaved DNA strand in a manner dictated by torsional strain in the DNA. In a second transesterification reaction, the nucleophilic attack by the 5Ј-OH resolves the covalent Top1-DNA intermediate, and the enzyme is liberated from the religated DNA.DNA cleavage by Top1p is, at least in part, rate-limiting, thus ensuring low steady state-levels of the covalent enzyme-DNA complex (4). However, increased concentrations of covalent complexes, either as a consequence of drug action, Top1p mutation, or the formation of DNA adducts, converts Top1p into a cellular poison (5-16). The camptothecin (CPT) class of chemotherapeutics reversibly stabilizes the covalent intermediat...

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“…Single amino acid substitutions in Top1p have also been defined that alter enzyme sensitivity to CPT and other Top1p poisons or that alter the DNA cleavage-religation equilibrium of the enzyme in the absence of drug (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). In yeast Top1T722Ap, substitution of Ala for Thr-722 (five residues N-terminal to the active site tyrosine, Tyr-727) mimics the action of CPT by reducing the rate of DNA religation (26,27).…”

mentioning

confidence: 99%

The Deubiquitinating Enzyme Doa4p Protects Cells from DNA Topoisomerase I Poisons

Fiorani

Reid

Schepis

et al. 2004

Journal of Biological Chemistry

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DNA topoisomerase I (Top1p) catalyzes changes in DNA topology via the formation of an enzyme-DNA covalent complex that is reversibly stabilized by the antitumor drug, camptothecin (CPT). During S-phase, collisions with replication forks convert these complexes into cytotoxic DNA lesions that trigger cell cycle arrest and cell death. To investigate cellular responses to CPT-induced DNA damage, a yeast genetic screen identified conditional tah mutants with enhanced sensitivity to self-poisoning DNA topoisomerase I mutant (Top1T722Ap), which mimics the action of CPT. Mutant alleles of three genes, DOA4, SLA1 and SLA2, were recovered. A nonsense mutation in DOA4 eliminated the catalytic residues of the Doa4p deubiquitinating enzyme, yet retained the rhodanase domain. At 36°C, this doa4-10 mutant exhibited increased sensitivity to CPT, osmotic stress, and hydroxyurea, and a reversible petite phenotype. However, the accumulation of pre-vacuolar class E vesicles that was observed in doa4⌬ cells was not detected in the doa4-10 mutant. Mutations in SLA1 or SLA2, which alter actin cytoskeleton architecture, induced a conditional synthetic lethal phenotype in combination with doa4-10 in the absence of DNA damage. Here actin cytoskeleton defects coincided with the enhanced fragility of large-budded cells. In contrast, the enhanced sensitivity of doa4-10 mutant cells to Top1T722Ap was unrelated to alterations in endocytosis and was selectively suppressed by increased dosage of the ribonucleotide reductase inhibitor Sml1p. Additional studies suggest a role for Doa4p in the Rad9p checkpoint response to Top1p poisons. These findings indicate a functional link between ubiquitin-mediated proteolysis and cellular resistance to CPT-induced DNA damage.DNA topoisomerases catalyze changes in the linkage of DNA strands, allowing for DNA unwinding or decatenation during DNA replication, transcription, recombination, and chromosome segregation (1, 2). Eukaryotic DNA topoisomerase I (Top1p) transiently cleaves a single strand of duplex DNA and forms a covalent tyrosyl linkage with a 3Ј-phosphoryl DNA end. The covalent Top1p-DNA intermediate allows for DNA rotation while conserving the energy of the cleaved phosphodiester bond.DNA topoisomerase I is the cellular target of the antitumor drug, camptothecin (CPT), 1 which reversibly stabilizes the covalent Top1p-DNA complex by inhibiting DNA religation (3-5). The ternary CPT-Top1p-DNA complexes per se are insufficient to induce a cytotoxic response. Rather, collisions with advancing replication forks convert the drug-stabilized complexes into the irreversible DNA lesions that trigger cell cycle arrest and cell death. This model is consistent with the S-phase specificity of CPT and the observation that inhibition of DNA synthesis by aphidicolin abrogates the cytotoxic action of the drug (6).In the yeast Saccharomyces cerevisiae, DNA topoisomerase

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Single Mutation in the Linker Domain Confers Protein Flexibility and Camptothecin Resistance to Human Topoisomerase I

Cited by 84 publications

References 32 publications

Pharmacogenetics of anticancer drug sensitivity in pancreatic cancer

Pharmacogenetics of anticancer drug sensitivity in pancreatic cancer

Mutation of Gly721 Alters DNA Topoisomerase I Active Site Architecture and Sensitivity to Camptothecin

The Deubiquitinating Enzyme Doa4p Protects Cells from DNA Topoisomerase I Poisons

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