Interaction between the N-terminus of human topoisomerase I and SV40 large T antigen.

We have attempted to identify human topoisomerase I-binding proteins in order to gain information regarding the cellular roles of this protein and the cytotoxic mechanisms of the anticancer drug camptothecin, which specifically targets topoisomerase I. In the course of this work we identified an interaction between the N-terminus of human topoisomerase I and the SV40 T antigen that is detectable in vitro using both affinity chromatography and co-immunoprecipitation. Additional results indicate that this interaction does not require intermediary DNA or stoichiometric quantities of other proteins. Furthermore, the interaction is detectable in vivo using a yeast two-hybrid assay. Two binding sites for T antigen are apparent on the topoisomerase I protein: one consisting of amino acids 1-139, the other present in the 383-765 region of the protein. Interestingly, nucleolin, which binds the 166-210 region of topoisomerase I, is able to bind an N-terminal fragment of topoisomerase I concurrently with T antigen. Taken together with our prior identification of nucleolin as a topoisomerase I-binding protein, the current results suggest that helicase-binding is a major role of the N-terminus of human topoisomerase I and that the resultant helicase-topoisomerase complex may function as a eukaryotic gyrase.     

Substitutions of Asn-726 in the active site of yeast DNA topoisomerase I define novel mechanisms of stabilizing the covalent enzyme-DNA intermediate

Eukaryotic DNA topoisomerase I (Top1p) catalyzes changes in DNA topology and is the cellular target of camptothecin. Recent reports of enzyme structure highlight the importance of conserved amino acids N-terminal to the active site tyrosine and the involvement of Asn-726 in mediating Top1p sensitivity to camptothecin. To investigate the contribution of this residue to enzyme catalysis, we evaluated the effect of substituting His, Asp, or Ser for Asn-726 on yeast Top1p. Top1N726S and Top1N726D mutant proteins were resistant to camptothecin, although the Ser mutant was distinguished by a lack of detectable changes in activity. Thus, a basic residue immediately N-terminal to the active site tyrosine is required for camptothecin cytotoxicity. However, replacing Asn-726 with Asp or His interfered with distinct aspects of the catalytic cycle, resulting in cell lethality. In contrast to camptothecin, which inhibits enzyme-catalyzed religation of DNA, the His substituent enhanced the rate of DNA scission, whereas the Asp mutation diminished the enzyme binding of DNA. Yet, these effects on enzyme catalysis were not mutually exclusive as the His mutant was hypersensitive to camptothecin. These results suggest distinct mechanisms of poisoning DNA topoisomerase I may be explored in the development of antitumor agents capable of targeting different aspects of the Top1p catalytic cycle.     

Active site mutations in DNA topoisomerase I distinguish the cytotoxic activities of camptothecin and the indolocarbazole, rebeccamycin.

DNA topoisomerase I (Top1p) catalyzes topological changes in DNA and is the cellular target of the antitumor agent camptothecin (CPT). Non-CPT drugs that target Top1p, such as indolocarbazoles, are under clinical development. However, whether the cytotoxicity of indolocarbazoles derives from Top1p poisoning remains unclear. To further investigate indolocarbazole mechanism, rebeccamycin R-3 activity was examined in vitro and in yeast. Using a series of Top1p mutants, where substitution of residues around the active site tyrosine has well-defined effects on enzyme catalysis, we show that catalytically active, CPT-resistant enzymes remain sensitive to R-3. This indolocarbazole did not inhibit yeast Top1p activity, yet was effective in stabilizing Top1p-DNA complexes. Similar results were obtained with human Top1p, when Ser or His were substituted for Asn-722. The mutations altered enzyme function and sensitivity to CPT, yet R-3 poisoning of Top1p was unaffected. Moreover, top1delta, rad52delta yeast cells expressing human Top1p, but not catalytically inactive Top1Y723Fp, were sensitive to R-3. These data support hTop1p as the cellular target of R-3 and indicate that distinct drug-enzyme interactions at the active site are required for efficient poisoning by R-3 or CPT. Furthermore, resistance to one poison may potentiate cell sensitivity to structurally distinct compounds that also target Top1p.     

Mitochondrial topoisomerase I sites in the regulatory D-loop region of mitochondrial DNA

Mitochondrial DNA (mtDNA) is required for mitochondrial activities because it encodes key proteins for oxidative phosphorylation and the production of cellular ATP. We previously reported the existence of a specific mitochondrial topoisomerase gene, Top1mt, in all vertebrates. The corresponding polypeptide contains an N-terminal mitochondrial targeting sequence and is otherwise highly homologous to the nuclear topoisomerase I (Top1). In this study, we provide biochemical evidence of the presence of an endogenous Top1mt polypeptide in human mitochondria. Using novel antibodies against Top1mt, we detected the corresponding 70 kDa polypeptide in mitochondria but not in nuclear fractions. This polypeptide could be trapped to form covalent complexes with mtDNA when mitochondria from human cells were treated with camptothecin. Mapping of Top1mt sites in the regulatory D-loop region of mtDNA in mitochondria revealed the presence of an asymmetric cluster of Top1mt sites confined to a 150 bp segment downstream from, and adjacent to, the site at which replication is prematurely terminated, generating an approximately 650-base (7S DNA) product that forms the mitochondrial D-loop. Moreover, we show that inhibition of Top1mt by camptothecin reduces the level of formation of the 7S DNA. These results suggest novel roles for Top1mt in regulating mtDNA replication.     

Identification of an N-terminal domain of eukaryotic DNA topoisomerase I dispensable for catalytic activity but essential for in vivo function.

We have found that deletion of a 70-amino acid domain, spanning from position 141 to 210 in the N-terminal part of human topoisomerase I, has no effect on the catalytic activity of the enzyme in vitro but suppresses the lethal consequence of overexpressing human topoisomerase I in a rad52 top1 Saccharomyces cerevisiae strain. By immunostaining, the 70-amino acid domain is shown to be necessary for nuclear location of topoisomerase I. We demonstrate that the nuclear localization signal from the SV40 large T antigen can substitute for the 70-amino acid domain, restoring both the lethal effect of overexpression and the correct subcellular localization of topoisomerase I. Thus, we have identified a domain in the N-terminal part of human topoisomerase I, nonessential for catalytic activity in vitro but serving an in vivo function by directing the enzyme to the nucleus. Based on sequence comparisons, we suggest that this domain is a conserved element in the apparently non-homologous N-terminal parts of yeast and human topoisomerase I.     

TDP2 promotes repair of topoisomerase I-mediated DNA damage in the absence of TDP1

The abortive activity of topoisomerases can result in clastogenic and/or lethal DNA damage in which the topoisomerase is covalently linked to the 3'- or 5'-terminus of a DNA strand break. This type of DNA damage is implicated in chromosome translocations and neurological disease and underlies the clinical efficacy of an important class of anticancer topoisomerase 'poisons'. Tyrosyl DNA phosphodiesterase-1 protects cells from abortive topoisomerase I (Top1) activity by hydrolyzing the 3'-phosphotyrosyl bond that links Top1 to a DNA strand break and is currently the only known human enzyme that displays this activity in cells. Recently, we identified a second tyrosyl DNA phosphodiesterase (TDP2; aka TTRAP/EAPII) that possesses weak 3'-tyrosyl DNA phosphodiesterase (3'-TDP) activity, in vitro. Herein, we have examined whether TDP2 contributes to the repair of Top1-mediated DNA breaks by deleting Tdp1 and Tdp2 separately and together in murine and avian cells. We show that while deletion of Tdp1 in wild-type DT40 cells and mouse embryonic fibroblasts decreases DNA strand break repair rates and cellular survival in response to Top1-induced DNA damage, deletion of Tdp2 does not. However, deletion of both Tdp1 and Tdp2 reduces rates of DNA strand break repair and cell survival below that observed in Tdp1(-)(/)(-) cells, suggesting that Tdp2 contributes to cellular 3'-TDP activity in the absence of Tdp1. Consistent with this idea, over-expression of human TDP2 in Tdp1(-)(/)(-)/Tdp2(-)(/)(-)(/)(-) DT40 cells increases DNA strand break repair rates and cell survival above that observed in Tdp1(-)(/)(-) DT40 cells, suggesting that Tdp2 over-expression can partially complement the defect imposed by loss of Tdp1. Finally, mice lacking both Tdp1 and Tdp2 exhibit greater sensitivity to Top1 poisons than do mice lacking Tdp1 alone, further suggesting that Tdp2 contributes to the repair of Top1-mediated DNA damage in the absence of Tdp1. In contrast, we failed to detect a contribution for Tdp1 to repair Top2-mediated damage. Together, our data suggest that Tdp1 and Tdp2 fulfil overlapping roles following Top1-induced DNA damage, but not following Top2-induced DNA damage, in vivo.     

NSR1 is required for pre-rRNA processing and for the proper maintenance of steady-state levels of ribosomal subunits.

NSR1 is a yeast nuclear localization sequence-binding protein showing striking similarity in its domain structure to nucleolin. Cells lacking NSR1 are viable but have a severe growth defect. We show here that NSR1, like nucleolin, is involved in ribosome biogenesis. The nsr1 mutant is deficient in pre-rRNA processing such that the initial 35S pre-rRNA processing is blocked and 20S pre-rRNA is nearly absent. The reduced amount of 20S pre-rRNA leads to a shortage of 18S rRNA and is reflected in a change in the distribution of 60S and 40S ribosomal subunits; there is no free pool of 40S subunits, and the free pool of 60S subunits is greatly increased in size. The lack of free 40S subunits or the improper assembly of these subunits causes the nsr1 mutant to show sensitivity to the antibiotic paromomycin, which affects protein translation, at concentrations that do not affect the growth of the wild-type strain. Our data support the idea that NSR1 is involved in the proper assembly of pre-rRNA particles, possibly by bringing rRNA and ribosomal proteins together by virtue of its nuclear localization sequence-binding domain and multiple RNA recognition motifs. Alternatively, NSR1 may also act to regulate the nuclear entry of ribosomal proteins required for proper assembly of pre-rRNA particles.     

The deubiquitinating enzyme Doa4p protects cells from DNA topoisomerase I poisons

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 degrees 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 doa4Delta 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.     

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 Ala(653) 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.     

Mutation of Gly721 alters DNA topoisomerase I active site architecture and sensitivity to camptothecin

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 anti-cancer 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 alpha-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 alpha-helix, which extends to the active site Tyr covalently linked to the DNA. In the presence of drug, the linker is rigid and this alpha-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 CPT-resistant. 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.     

Yeast NSR1 protein that has structural similarity to mammalian nucleolin is involved in pre-rRNA processing.

We have identified a yeast gene encoding a protein structurally similar to mammalian nucleolin. The gene was previously cloned as a cold shock-inducible gene and found to be identical to yeast NSR1 gene, which encodes a protein that has been reported to bind sequences required for nuclear localization of protein. The carboxyl-terminal half of NSR1, consisting of two tandemly repeated putative RNA-binding domains and a glycine/arginine-rich domain, has 37% amino acid sequence identity with the same part of mammalian nucleolin, while no sequence similarities are found between their amino-terminal regions. Although a null mutation of the NSR1 gene was not lethal, it caused a severe defect on growth. Pulse-labeling analysis revealed that the nsr1 strain had reduced levels of 18 S rRNA and accumulated 35 S pre-rRNA compared with the wild-type strain. The level of 25 S rRNA was also slightly reduced in the nsr1 strain. Pulse-chase labeling experiments showed slow processing of 35 S pre-rRNA and impaired methylation of 18 S rRNA. The ratio of 40 S to 60 S ribosomal subunits in the nsr1 strain is significantly reduced and is consistent with impaired synthesis of 18 S rRNA. The results indicate that NSR1 is involved in pre-rRNA processing and ribosome biosynthesis in yeast.     

Box C/D small nucleolar RNA trafficking involves small nucleolar RNP proteins, nucleolar factors and a novel nuclear domain

Nucleolar localization of box C/D small nucleolar (sno) RNAs requires the box C/D motif and, in vertebrates, involves transit through Cajal bodies (CB). We report that in yeast, overexpression of a box C/D reporter leads to a block in the localization pathway with snoRNA accumulation in a specific sub-nucleolar structure, the nucleolar body (NB). The human survival of motor neuron protein (SMN), a marker of gems/CB, specifically localizes to the NB when expressed in yeast, supporting similarities between these structures. Box C/D snoRNA accumulation in the NB was decreased by mutation of Srp40 and increased by mutation of Nsr1p, two related nucleolar proteins that are homologous to human Nopp140 and nucleolin, respectively. Box C/D snoRNAs also failed to accumulate in the NB, and became delocalized to the nucleoplasm, upon depletion of any of the core snoRNP proteins, Nop1p/fibrillarin, Snu13p, Nop56p and Nop5p/Nop58p. We conclude that snoRNP assembly occurs either in the nucleoplasm, or during transit of snoRNAs through the NB, followed by routing of the complete snoRNP to functional sites of ribosome synthesis.     

Residues within the N-terminal domain of human topoisomerase I play a direct role in relaxation

All eukaryotic forms of DNA topoisomerase I contain an extensive and highly charged N-terminal domain. This domain contains several nuclear localization sequences and is essential for in vivo function of the enzyme. However, so far no direct function of the N-terminal domain in the in vitro topoisomerase I reaction has been reported. In this study we have compared the in vitro activities of a truncated form of human topoisomerase I lacking amino acids 1-206 (p67) with the full-length enzyme (p91). Using these enzyme forms, we have identified for the first time a direct role of residues within the N-terminal domain in modulating topoisomerase I catalysis, as revealed by significant differences between p67 and p91 in DNA binding, cleavage, strand rotation, and ligation. A comparison with previously published studies showing no effect of deleting the first 174 or 190 amino acids of topoisomerase I (Stewart, L., Ireton, G. C., and Champoux, J. J. (1999) J. Biol. Chem. 274, 32950-32960; Bronstein, I. B., Wynne-Jones, A., Sukhanova, A., Fleury, F., Ianoul, A., Holden, J. A., Alix, A. J., Dodson, G. G., Jardillier, J. C., Nabiev, I., and Wilkinson, A. J. (1999) Anticancer Res. 19, 317-327) suggests a pivotal role of amino acids 191-206 in catalysis. Taken together the presented data indicate that at least part(s) of the N-terminal domain regulate(s) enzyme/DNA dynamics during relaxation most probably by controlling non-covalent DNA binding downstream of the cleavage site either directly or by coordinating DNA contacts by other parts of the enzyme.     

In vivo analysis of nucleolar proteins modified by the yeast arginine methyltransferase Hmt1/Rmt1p

In this report, we have investigated the impact of arginine methylation on the Gar1, Nop1, and Nsr1 nucleolar proteins in Saccharomyces cerevisiae. Although previous reports have established that protein arginine methylation is important for nucleocytoplasmic shuttling, they have focused on the examination of heterogeneous nuclear ribonucleoproteins (hnRNPs). We have extended this analysis to several nucleolar proteins that represent a distinct functional class of arginine-methylated proteins. We first developed an in vivo assay to identify proteins methylated by the Hmt1 arginine methyltransferase. This assay is based on the fact that the Hmt1 enzyme utilizes S-Adenosyl-L-methionine as the methyl donor for protein arginine methylation. Following SDS polyacrylamide electrophoresis, 11 distinct proteins were identified as substrates for the Hmt1 methyltransferase. Hmt1p overexpression did not increase the methylation level on these proteins, suggesting they are fully methylated under the conditions examined. Three of the radiolabeled proteins were confirmed to be Gar1p, Nop1p, and Nsr1p. To monitor the cellular localization of these proteins, functional GFP fusion proteins were generated and found to be localized to the nucleolus. This localization was independent of arginine methylation. Furthermore, all three proteins examined did not export to the cytoplasm. In contrast, arginine methylation is required for the export of the nuclear RNA-binding proteins Npl3p, Hrp1p, and Nab2p. The observation that three nucleolar proteins are modified by Hmt1p but are not exported from the nucleolus implies an alternate role for arginine methylation.     

Serine phosphorylation-dependent coregulation of topoisomerase I by the p14ARF tumor suppressor

p14ARF (ARF) and topoisomerase I play central roles in cancer and have recently been shown to interact. The interaction activates topoisomerase I, an important target for camptothecin-like chemotherapeutic drugs, but the regulation of the interaction is poorly understood. We have used the H358 and H23 lung cancer cell lines and purified recombinant human topoisomerase I to demonstrate that the ARF/topoisomerase I interaction is regulated by topoisomerase I serine phosphorylation, a modification that regulates topoisomerase I activity. Both cell lines express wild-type ARF and topoisomerase I proteins at equivalent levels, but H23 topoisomerase I, unlike that of H358 cells, is largely devoid of serine phosphorylation, has low activity, and complexes poorly with ARF. The ability of H23 topoisomerase I to complex with ARF can be restored by treatment with the serine kinase, casein kinase II. Consistent with these observations, we show that the response of H23 cells to camptothecin treatment is unaffected by changes in intracellular levels of ARF. However, in H358 and PC-3 cells, which express a serine phosphorylated topoisomerase I that complexes with ARF, ectopic overexpression of ARF causes sensitization to camptothecin, and siRNA-mediated down-regulation of endogenous ARF causes desensitization to camptothecin. These biological responses correlate with increased and decreased levels, respectively, of ARF/topoisomerase I complex and DNA-bound topoisomerase I. Thus, ARF is a serine phosphorylation-dependent coregulator of topoisomerase I in vivo, and it regulates cellular sensitivity to camptothecin by interacting with topoisomerase I. Certain cancer associated defects affecting ARF/topoisomerase I complex formation could contribute to cellular resistance to camptothecin.     

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-clamp(534) 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-clamp(499) 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.