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.     

Substitution of conserved residues within the active site alters the cleavage religation equilibrium of DNA topoisomerase I

Eukaryotic DNA topoisomerase I (Top1p) catalyzes the relaxation of supercoiled DNA and constitutes the cellular target of camptothecin (CPT). Mutation of conserved residues in close proximity to the active site tyrosine (Tyr(727) of yeast Top1p) alters the DNA cleavage religation equilibrium, inducing drug-independent cell lethality. Previous studies indicates that yeast Top1T722Ap and Top1N726Hp cytotoxicity results from elevated levels of covalent enzyme-DNA intermediates. Here we show that Top1T722Ap acts as a CPT mimetic by exhibiting reduced rates of DNA religation, whereas increased Top1N726Hp.DNA complexes result from elevated DNA binding and cleavage. We also report that the combination of the T722A and N726H mutations in a single protein potentiates the cytotoxic action of the enzyme beyond that induced by co-expression of the single mutants. Moreover, the addition of CPT to cells expressing the double top1T722A/N726H mutant did not enhance cell lethality. Thus, independent alterations in DNA cleavage and religation contribute to the lethal phenotype. The formation of distinct cytotoxic lesions was also evidenced by the different responses induced by low levels of these self-poisoning enzymes in isogenic strains defective for the Rad9 DNA damage checkpoint, processive DNA replication, or ubiquitin-mediated proteolysis. Substitution of Asn(726) with Phe or Tyr also produces self-poisoning enzymes, implicating stacking interactions in the increased kinetics of DNA cleavage by Top1N726Hp and Top1N726Fp. In contrast, replacing the amide side chain of Asn(726) with Gln renders Top1N726Qp resistant to CPT, suggesting that the orientation of the amide within the active site is critical for effective CPT binding.     

Ubc9 is required for damage-tolerance and damage-induced interchromosomal homologous recombination in S. cerevisiae

Ubc9 is an enzyme involved in the conjugation of small ubiquitin related modifier (SUMO) to target proteins. A Saccharomyces cerevisiae ubc9 temperature sensitive (ts) mutant showed higher sensitivity to various DNA damaging agents such as methylmethanesulfonate (MMS) and UV at a semi-permissive temperature than wild-type cells. The sensitivity of ubc9ts cells was not suppressed by the introduction of a mutated UBC9 gene, UBC9-C93S, whose product is unable to covalently bind to SUMO and consequently fails to conjugate SUMO to target proteins. Diploid ubc9ts cells were more sensitive to various DNA damaging agents than haploid ubc9ts cells suggesting the involvement of homologous recombination in the sensitivity of ubc9ts cells. The frequency of interchromosomal recombination between heteroalleles, his1-1/his1-7 loci, in wild-type cells was remarkably increased upon exposure to MMS or UV. Although the frequency of spontaneous interchromosomal recombination between the heteroalleles in ubc9ts cells was almost the same as that of wild-type cells, no induction of interchromosomal recombination was observed in ubc9ts cells upon exposure to MMS or UV.     

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.     

Platinated DNA adducts enhance poisoning of DNA topoisomerase I by camptothecin

Camptothecins constitute a novel class of chemotherapeutics that selectively target DNA topoisomerase I (Top1) by reversibly stabilizing a covalent enzyme-DNA intermediate. This cytotoxic mechanism contrasts with that of platinum drugs, such as cisplatin, which induce inter- and intrastrand DNA adducts. In vitro combination studies using platinum drugs combined with Top1 poisons, such as topotecan, showed a schedule-dependent synergistic activity, with promising results in the clinic. However, whereas the molecular mechanism of these single agents may be relatively well understood, the mode of action of these chemotherapeutic agents in combination necessitates a more complete understanding. Indeed, we recently reported that a functional homologous recombination pathway is required for cisplatin and topotecan synergy yet represses the synergistic toxicity of 1-beta-D-arabinofuranosyl cytidine in combination with topotecan (van Waardenburg, R. C., de Jong, L. A., van Delft, F., van Eijndhoven, M. A., Bohlander, M., Bjornsti, M. A., Brouwer, J., and Schellens, J. H. (2004) Mol. Cancer Ther. 3, 393-402). Here we provide direct evidence for Pt-1,3-d(GTG) poisoning of Top1 in vitro and demonstrate that persistent Pt-DNA adducts correlate with increased covalent Top1-DNA complexes in vivo. This contrasts with a lack of persistent lesions induced by the alkylating agent bis[chloroethyl]nitrosourea, which exhibits only additive activity with topotecan in a range of cell lines. In human IGROV-1 ovarian cancer cells, the synergistic activity of cisplatin with topotecan requires processive DNA polymerization, whereas overexpression of Top1 enhances yeast cell sensitivity to cisplatin. These results indicate that the cytotoxic activity of cisplatin is due, in part, to poisoning of Top1, which is exacerbated in the presence of topotecan.     

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.     

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.     

Topoisomerase III acts upstream of Rad53p in the S-phase DNA damage checkpoint

Deletion of the Saccharomyces cerevisiae TOP3 gene, encoding Top3p, leads to a slow-growth phenotype characterized by an accumulation of cells with a late S/G2 content of DNA (S. Gangloff, J. P. McDonald, C. Bendixen, L. Arthur, and R. Rothstein, Mol. Cell. Biol. 14:8391-8398, 1994). We have investigated the function of TOP3 during cell cycle progression and the molecular basis for the cell cycle delay seen in top3Delta strains. We show that top3Delta mutants exhibit a RAD24-dependent delay in the G2 phase, suggesting a possible role for Top3p in the resolution of abnormal DNA structures or DNA damage arising during S phase. Consistent with this notion, top3Delta strains are sensitive to killing by a variety of DNA-damaging agents, including UV light and the alkylating agent methyl methanesulfonate, and are partially defective in the intra-S-phase checkpoint that slows the rate of S-phase progression following exposure to DNA-damaging agents. This S-phase checkpoint defect is associated with a defect in phosphorylation of Rad53p, indicating that, in the absence of Top3p, the efficiency of sensing the existence of DNA damage or signaling to the Rad53 kinase is impaired. Consistent with a role for Top3p specifically during S phase, top3Delta mutants are sensitive to the replication inhibitor hydroxyurea, expression of the TOP3 mRNA is activated in late G1 phase, and DNA damage checkpoints operating outside of S phase are unaffected by deletion of TOP3. All of these phenotypic consequences of loss of Top3p function are at least partially suppressed by deletion of SGS1, the yeast homologue of the human Bloom's and Werner's syndrome genes. These data implicate Top3p and, by inference, Sgs1p in an S-phase-specific role in the cellular response to DNA damage. A model proposing a role for these proteins in S phase is presented.     

Sumoylation of amyloid precursor protein negatively regulates Abeta aggregate levels

The proteolytic processing of amyloid precursor protein (APP) to produce Aβ peptides is thought to play an important role in the mechanism of Alzheimer’s disease. Here, we show that lysines 587 and 595 of APP, which are immediately adjacent to the site of β-secretase cleavage, are covalently modified by SUMO proteins in vivo. Sumoylation of these lysine residues is associated with decreased levels of Aβ aggregates. Further, overexpression of the SUMO E2 enzyme ubc9 along with SUMO-1 results in decreased levels of Aβ aggregates in cells transfected with the familial Alzheimer’s disease-associated V642F mutant APP, indicating the potential of up-regulating activity of the cellular sumoylation machinery as an approach against Alzheimer’s disease. The results also provide the first demonstration that the SUMO E2 enzyme (ubc9) is present within the endoplasmic reticulum, indicating how APP, and perhaps other proteins that enter this compartment, can be sumoylated.     

Cooperation of sumoylated chromosomal proteins in rDNA maintenance

SUMO is a posttranslational modifier that can modulate protein activities, interactions, and localizations. As the GFP-Smt3p fusion protein has a preference for subnucleolar localization, especially when deconjugation is impaired, the nucleolar role of SUMO can be the key to its biological functions. Using conditional triple SUMO E3 mutants, we show that defects in sumoylation impair rDNA maintenance, i.e., the rDNA segregation is defective and the rDNA copy number decreases in these mutants. Upon characterization of sumoylated proteins involved in rDNA maintenance, we established that Top1p and Top2p, which are sumoylated by Siz1p/Siz2p, most likely collaborate with substrates of Mms21p to maintain rDNA integrity. Cohesin and condensin subunits, which both play important roles in rDNA stability and structures, are potential substrates of Mms21, as their sumoylation depends on Mms21p, but not Siz1p and Siz2p. In addition, binding of cohesin and condensin to rDNA is altered in the mms21-CH E3-deficient mutant.     

Topoisomerase I-dependent viability loss in saccharomyces cerevisiae mutants defective in both SUMO conjugation and DNA repair

Siz1 and Siz2/Nfi1 are the two Siz/PIAS SUMO E3 ligases in Saccharomyces cerevisiae. Here we show that siz1Delta siz2Delta mutants fail to grow in the absence of the homologous recombination pathway or the Fen1 ortholog RAD27. Remarkably, the growth defects of mutants such as siz1Delta siz2Delta rad52Delta are suppressed by mutations in TOP1, suggesting that these growth defects are caused by topoisomerase I activity. Other mutants that affect SUMO conjugation, including a ulp1 mutant and the nuclear pore mutants nup60Delta and nup133Delta, show similar top1-suppressible synthetic defects with DNA repair mutants, suggesting that these phenotypes also result from reduced SUMO conjugation. siz1Delta siz2Delta mutants also display TOP1-independent genome instability phenotypes, including increased mitotic recombination and elongated telomeres. We also show that SUMO conjugation, TOP1, and RAD27 have overlapping roles in telomere maintenance. Top1 is sumoylated, but Top1 does not appear to be the SUMO substrate involved in the synthetic growth defects. However, sumoylation of certain substrates, including Top1 itself and Tri1 (YMR233W), is enhanced in the absence of Top1 activity. Sumoylation is also required for growth of top1Delta cells. These results suggest that the SUMO pathway has a complex effect on genome stability that involves several mechanistically distinct processes.     

Enhanced CPT sensitivity of yeast cells and selective relaxation of Ga14 motif-containing DNA by novel Gal4-topoisomerase I fusion proteins

Human topoisomerase I-B (Top1) efficiently relaxes DNA supercoils during basic cellular processes, and can be transformed into a DNA-damaging agent by antitumour drugs, enzyme mutations and DNA lesions. Here, we describe Gal4-Top1 chimeric proteins (GalTop) with an N-terminal truncation of Top1, and mutations of the Gal4 Zn-cluster and/or Top1 domains that impair their respective DNA-binding activities. Expression levels of chimeras were similar in yeast cells, however, GalTop conferred an increased CPT sensitivity to RAD52- yeast cells as compared to a GalTop with mutations of the Gal4 domain, showing that a functional Gal4 domain can alter in vivo functions of Top1. In vitro enzyme activity was tested with a DNA relaxation assay using negatively supercoiled plasmids with 0 to 5 Gal4 consensus motifs. Only GalTop with a functional Gal4 domain could direct DNA relaxation activity of Top1 specifically to DNA molecules containing Gal4 motifs. By using a substrate competition assay, we could demonstrate that the Gal4-anchored Top1 remains functional and efficiently relax DNA substrates in cis. The enhanced CPT sensitivity of GalTop in yeast cells may then be due to alterations of the chromatin-binding activity of Top1. The GalTop chimeras may indeed mimic a normal mechanism by which Top1 is recruited to chromatin sites in living cells. Such hybrid Top1s may be helpful in further dissecting enzyme functions, and constitute a prototype of a site-specific DNA cutter endowed with high cell lethality.     

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.     

Association of XRCC1 and tyrosyl DNA phosphodiesterase (Tdp1) for the repair of topoisomerase I-mediated DNA lesions

DNA topoisomerase I (Top1) is converted into a cellular poison by camptothecin (CPT) and various endogenous and exogenous DNA lesions. In this study, we used X-ray repair complementation group 1 (XRCC1)-deficient and XRCC1-complemented EM9 cells to investigate the mechanism by which XRCC1 affects the cellular responses to Top1 cleavage complexes induced by CPT. XRCC1 complementation enhanced survival to CPT-induced DNA lesions produced independently of DNA replication. CPT-induced comparable levels of Top1 cleavage complexes (single-strand break (SSB) and DNA-protein cross-links (DPC)) in both XRCC1-deficient and XRCC1-complemented cells. However, XRCC1-complemented cells repaired Top1-induced DNA breaks faster than XRCC1-deficient cells, and exhibited enhanced tyrosyl DNA phosphodiesterase (Tdp1) and polynucleotide kinase phosphatase (PNKP) activities. XRCC1 immunoprecipitates contained Tdp1 polypeptide, and both Tdp1 and PNKP activities, indicating a functional connection between the XRCC1 single-strand break repair pathway and the repair of Top1 covalent complexes by Tdp1 and PNKP.     

Topoisomerase IIβ Deficiency Enhances Camptothecin-induced Apoptosis

Camptothecin (CPT), a topoisomerase (Top) I-targeting drug that stabilizes Top1-DNA covalent adducts, can induce S-phase-specific cytotoxicity due to the arrest of progressing replication forks. However, CPT-induced non-S-phase cytotoxicity is less well characterized. In this study, we have identified topoisomerase IIβ (Top2β) as a specific determinant for CPT sensitivity, but not for many other cytotoxic agents, in non-S-phase cells. First, quiescent mouse embryonic fibroblasts (MEFs) lacking Top2β were shown to be hypersensitive to CPT with prominent induction of apoptosis. Second, ICRF-187, a Top2 catalytic inhibitor known to deplete Top2β, specifically sensitized MEFs to CPT. To explore the molecular basis for CPT hypersensitivity in Top2β-deficient cells, we found that upon CPT exposure, the RNA polymerase II large subunit (RNAP LS) became progressively depleted, followed by recovery to nearly the original level in wild-type MEFs, whereas RNAP LS remained depleted without recovery in Top2β-deficient cells. Concomitant with the reduction of the RNAP LS level, the p53 protein level was greatly induced. Interestingly, RNAP LS depletion has been well documented to lead to p53-dependent apoptosis. Altogether, our findings support a model in which Top2β deficiency promotes CPT-induced apoptosis in quiescent non-S-phase cells, possibly due to RNAP LS depletion and p53 accumulation.