Piero Benedetti

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

DNA topoisomerase I relaxes supercoiled DNA by the formation of a covalent intermediate in which the active-site tyrosine is transiently bound to the cleaved DNA strand. The antineoplastic agent camptothecin specifically targets DNA topoisomerase I, and several mutations have been isolated that render the enzyme camptothecin-resistant. The catalytic and structural dynamical properties of a human DNA topoisomerase I mutant in which Ala-653 in the linker domain was mutated into Pro have been investigated. The mutant is resistant to camptothecin and in the absence of the drug displays a cleavage-religation equilibrium strongly shifted toward religation. The shift is mainly because of an increase in the religation rate relative to the wild type enzyme, indicating that the unperturbed linker is involved in slowing religation. Molecular dynamics simulation indicates that the Ala to Pro mutation increases the linker flexibility allowing it to sample a wider conformational space. The increase in religation rate of the mutant, explained by means of the enhanced linker flexibility, provides an explanation for the mutant camptothecin resistance.

Yeast as a model organism for studying the actions of DNA topoisomerase-targeted drugs

The budding yeast Saccharomyces cerevisiae has been exploited to investigate the cytotoxic mechanisms of drugs that target DNA topoisomerases. This model organism has been used to establish eukaryotic DNA topoisomerase I or II as the cellular target of specific antineoplastic agents, to define mutations in these enzymes that confer drug resistance and to elucidate the cellular factors that modulate cell sensitivity to DNA topoisomerase-targeted drugs. These findings have provided valuable insights into the critical activities of these enzymes and how perturbing their functions produces DNA damage and cell death.

Thr729 in human topoisomerase I modulates anti-cancer drug resistance by altering protein domain communications as suggested by molecular dynamics simulations

The role of Thr729 in modulating the enzymatic function of human topoisomerase I has been characterized by molecular dynamics (MD) simulation. In detail, the structural–dynamical behaviour of the Thr729Lys and the Thr729Pro mutants have been characterized because of their in vivo and in vitro functional properties evidenced in the accompanying paper. Both mutants can bind to the DNA substrate and are enzymatically active, but while Thr729Lys is resistant even at high concentration of the camptothecin (CPT) anti-cancer drug, Thr729Pro shows only a mild reduction in drug sensitivity and in DNA binding. MD simulations show that the Thr729Lys mutation provokes a structural perturbation of the CPT-binding pocket. On the other hand, the Thr729Pro mutant maintains the wild-type structural scaffold, only increasing its rigidity. The simulations also show the complete abolishment, in the Thr729Lys mutant, of the protein communications between the C-terminal domain (where the active Tyr723 is located) and the linker domain, that plays an essential role in the control of the DNA rotation, thus explaining the distributive mode of action displayed by this mutant.

Locking the DNA topoisomerase I protein clamp inhibits DNA rotation and induces cell lethality.

Eukaryotic DNA topoisomerase I (Top1) is a monomeric protein clamp that functions in DNA replication, transcription, and recombination. Opposable “lip” domains form a salt bridge to complete Top1 protein clamping of duplex DNA. Changes in DNA topology are catalyzed by the formation of a transient phosphotyrosyl linkage between the active-site Tyr-723 and a single DNA strand. Substantial protein domain movements are required for DNA binding, whereas the tight packing of DNA within the covalent Top1–DNA complex necessitates some DNA distortion to allow rotation. To investigate the effects of Top1-clamp closure on enzyme catalysis, molecular modeling was used to design a disulfide bond between residues Gly-365 and Ser-534, to crosslink protein loops more proximal to the active-site tyrosine than the protein loops held by the Lys-369–Glu-497 salt bridge. In reducing environments, Top1-Clamp was catalytically active. However, contrary to crosslinking the salt-bridge loops [Carey, J. F., Schultz, S. J., Sission, L., Fazzio, T. G. & Champoux, J. J. (2003) Proc. Natl. Acad. Sci. USA 100, 5640–5645], crosslinking the active-site proximal loops inhibited DNA rotation. Apparently, subtle alterations in Top1 clamp flexibility impact enzyme catalysis in vitro. Yet, the catalytically active Top1-Clamp was cytotoxic, even in the reducing environment of yeast cells. Remarkably, a shift in redox potential in glr1Δ cells converted the catalytically inactive Top1Y723F mutant clamp into a cellular toxin, which failed to induce an S-phase terminal phenotype. This cytotoxic mechanism is distinct from that of camptothecin chemotherapeutics, which stabilize covalent Top1–DNA complexes, and it suggests that the development of novel therapeutics that promote Top1-clamp closure is possible.

Effect on DNA relaxation of the single Thr718Ala mutation in human topoisomerase I: a functional and molecular dynamics study

The functional and dynamical properties of the human topoisomerase I Thr718Ala mutant have been compared to that of the wild-type enzyme using functional assays and molecular dynamics (MD) simulations. At physiological ionic strength, the cleavage and religation rates, evaluated on oligonucleotides containing the preferred topoisomerase I DNA sequence, are almost identical for the wild-type and the mutated enzymes, as is the cleavage/religation equilibrium. On the other hand, the Thr718Ala mutant shows a decreased efficiency in a DNA plasmid relaxation assay. The MD simulation, carried out on the enzyme complexed with its preferred DNA substrate, indicates that the mutant has a different dynamic behavior compared to the wild-type enzyme. Interestingly, no changes are observed in the proximity of the mutation site, whilst a different flexibility is detected in regions contacting the DNA scissile strand, such as the linker and the V-shaped α helices. Taken together, the functional and simulation results indicate a direct communication between the mutation site and regions located relatively far away, such as the linker domain, that with their altered flexibility confer a reduced DNA relaxation efficiency. These results provide evidence that the comprehension of the topoisomerase I dynamical properties are an important element in the understanding of its complex catalytic cycle.

YeastSaccharomyces cerevisiae as a model system to study the cytotoxic activity of the antitumor drug camptothecin

Eukaryotic DNA topoisomerase I catalyzes the relaxation of positively and negatively supercoiled DNA and plays a critical role in processes involving DNA, such as DNA replication, transcription and recombination. The enzyme is encoded by theTOP1 gene and is highly conserved in its amino acid sequence and sensitivity to the anti-neoplatic agent camptothecin. This plant alkaloid specifically targets DNA topoisomerase I by reversibly stabilizing the covalent enzyme-DNA intermediate. Presumably, it is the interaction of these drug-stabilized adducts with other cellular components, such as replication forks, that actually produces the DNA lesions leading to cell death. A conservation of the mechanism(s) of camptothecin-induced cell killing is also implicit in studies of the yeastSaccharomyces cerevisiae, where the camptothecin sensitivity of ΔTOP1 yeast cells can be restored by plasmids expressing either yeast or humanTOP1 sequences. This genetically tractable system is currently being exploited to describe the specific molecular interactions required for the cytotoxic action of camptothecin. The results of mutational analyses of yeast and human DNA topoisomerase I are presented, as well as a genetic screen designed to identify genes, other thanTOP1, that are required for the cytotoxic activity of camptothecin.

Alterations in linker flexibility suppress DNA topoisomerase I mutant-induced cell lethality

Eukaryotic DNA topoisomerase I (Top1p) catalyzes changes in DNA topology via the formation of a covalent enzyme-DNA intermediate, which is reversibly stabilized by the anticancer agent camptothecin (CPT). Crystallographic studies of the 70-kDa C terminus of human Top1p bound to duplex DNA describe a monomeric protein clamp circumscribing the DNA helix. The structures, which lack the N-terminal domain, comprise the conserved clamp, an extended linker domain, and the conserved C-terminal active site Tyr domain. CPT bound to the covalent Top1p-DNA complex limits linker flexibility, allowing structural determination of this domain. We previously reported that mutation of Ala653 to Pro in the linker increases the rate of enzyme-catalyzed DNA religation, thereby rendering Top1A653Pp resistant to CPT (Fiorani, P., Bruselles, A., Falconi, M., Chillemi, G., Desideri, A., and Benedetti P. (2003) J. Biol. Chem. 278, 43268–43275). Molecular dynamics studies suggested mutation-induced increases in linker flexibility alter Top1p catalyzed DNA religation. To address the functional consequences of linker flexibility on enzyme catalysis and drug sensitivity, we investigated the interactions of the A653P linker mutation with a self-poisoning T718A mutation within the active site of Top1p. The A653P mutation suppressed the lethal phenotype of Top1T718Ap in yeast, yet did not restore enzyme sensitivity to CPT. However, the specific activity of the double mutant was decreased in vivo and in vitro, consistent with a decrease in DNA binding. These findings support a model where changes in the flexibility or orientation of the linker alter the geometry of the active site and thereby the kinetics of DNA cleavage/religation catalyzed by Top1p.

A single mutation in the 729 residue modulates human DNA topoisomerase IB DNA binding and drug resistance

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.

Disulfide Cross-links Reveal Conserved Features of DNA Topoisomerase I Architecture and a Role for the N Terminus in Clamp Closure

In eukaryotes, DNA topoisomerase I (Top1) catalyzes the relaxation of supercoiled DNA by a conserved mechanism of transient DNA strand breakage, rotation, and religation. The unusual architecture of the monomeric human enzyme comprises a conserved protein clamp, which is tightly wrapped about duplex DNA, and an extended coiled-coil linker domain that appropriately positions the C-terminal active site tyrosine domain against the Top1 core to form the catalytic pocket. A structurally undefined N-terminal domain, dispensable for enzyme activity, mediates protein-protein interactions. Previously, reversible disulfide bonds were designed to assess whether locking the Top1 clamp around duplex DNA would restrict DNA strand rotation within the covalent Top1-DNA intermediate. The active site proximal disulfide bond in full-length Top1-clamp534 restricted DNA rotation (Woo, M. H., Losasso, C., Guo, H., Pattarello, L., Benedetti, P., and Bjornsti, M. A. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 13767–13772), whereas the more distal disulfide bond of the N-terminally truncated Topo70-clamp499 did not (Carey, J. F., Schultz, S. J., Sisson, L., Fazzio, T. G., and Champoux, J. J. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 5640–5645). To assess the contribution of the N-terminal domain to the dynamics of Top1 clamping of DNA, the same disulfide bonds were engineered into full-length Top1 and truncated Topo70, and the activities of these proteins were assessed in vitro and in yeast. Here we report that the N terminus impacts the opening and closing of the Top1 protein clamp. We also show that the architecture of yeast and human Top1 is conserved in so far as cysteine substitutions of the corresponding residues suffice to lock the Top1-clamp. However, the composition of the divergent N-terminal/linker domains impacts Top1-clamp activity and stability in vivo.

Analysis of comptothecin resistance in yeast: relevance to cancer therapy

The budding yeast Saccharomyces cerevisiae is a well defined genetic system to investigate various aspects of camptothecin (Cpt)-induced cytotoxicity. This antineoplastic agent and its derivatives specifically poison eukaryotic DNA topoisomerase I, the product of the TOP1 gene, by stabilizing a covalent enzyme-DNA intermediate. Analyses of various yeast and human top1 mutants in yeast strains deleted for TOP1 (top1Delta) have defined amino acid residues critical for enzyme function and Cpt sensitivity. Cpt cytotoxicity is also mediated by the pleiotropic drug resistance network, primarily through the action of an ABC transporter. The potential clinical relevance of these and related studies are discussed.