Topoisomerases are ubiquitous enzymes that control DNA supercoiling and entanglements. They are essential during transcription and replication and topoisomerase inhibitors are among the most effective and most commonly used anticancer and antibacterial drugs. This review consists in two parts. In the first part (“Lessons”), it gives background information on the catalytic mechanisms of the different enzyme families (6 different genes in humans and 4 in most bacteria), describes the “interfacial inhibition” by which topoisomerase-targeted drugs act as topoisomerase poisons and describes clinically relevant topoisomerase inhibitors. It generalizes the interfacial inhibition principle, which was discovered from the mechanism of action of topoisomerase inhibitors, and discusses how topoisomerase inhibitors kill cells by trapping topoisomerases on DNA rather than by classical enzymatic inhibition. Trapping protein-DNA complexes extends to a novel mechanism of action of PARP inhibitors and could be applied to the targeting of transcription factors. The second part of the review focuses on the challenges for discovery and precise use of topoisomerase inhibitors, including targeting topoisomerase inhibitors using chemical coupling and encapsulation for selective tumor delivery, use of pharmacodynamic biomarkers to follow drug activity, complexity of the response determinants for anticancer activity and patient selection, prospects of rational combinations with DNA repair inhibitors targeting tyrosyl-DNA-phosphodiesterases 1 and 2 (TDP1 and TDP2) and PARP, and the unmeet need to develop inhibitors for type IA enzymes.
Transcription has the capacity to modify mechanically DNA topology, DNA structure, and nucleosome arrangement. Resulting from ongoing transcription, these modifications in turn, may provide instant feedback to the transcription machinery. To substantiate the connection between transcription and DNA dynamics, we charted an ENCODE map of transcription-dependent dynamic supercoiling in human Burkitt lymphoma cells using psoralen photobinding to probe DNA topology in vivo. Dynamic supercoils spread ~1.5 kb upstream of the start sites of active genes. Low and high output promoters handle this torsional stress differently as shown using inhibitors of transcription and topoisomerases, and by chromatin immunoprecipation of RNA polymerase and topoisomerases I and II. Whereas lower outputs are managed adequately by topoisomerase I, high output promoters additionally require topoisomerase II. The genome-wide coupling between transcription and DNA topology emphasizes the importance of dynamic supercoiling for gene regulation.
The role of DNA topoisomerases in eucaryotic class III gene transcription in vitro has been studied through the use of inhibitory drugs and antisera to DNA topoisomerases I and II. The DNA topoisomerase II inhibitors, novobiocin and coumermycin AI, were found to inhibit transcription of cloned 5S and tRNA genes. Novobiocin acts by interfering with an ATP-requiring step in the pathway to stable preinitiation complex formation. However, it is unlikely that this step reflects the enzymatic action of DNA topoisomerase II since a specific inhibitor of this enzyme (VM-26) and anti-DNA topoisomerase II antibodies fail to inhibit transcription under conditions where topoisomerase II enzymatic activity is inhibited. Similarly, a specific inhibitor of DNA topoisomerase I (camptothecin) and anti-DNA topoisomerase I antibodies fail to inhibit class III gene transcription. These results argue against a role for either DNA topoisomerase in 5S or tRNA gene transcription in vitro.
Replication forks stalled by excess DNA supercoiling can be resolved by DNA cleavage by the Mus81 endonuclease.
Deoxyribonucleic acid (DNA) topoisomerases are essential for removing the supercoiling that normally builds up ahead of replication forks. The camptothecin (CPT) Top1 (topoisomerase I) inhibitors exert their anticancer activity by reversibly trapping Top1–DNA cleavage complexes (Top1cc’s) and inducing replication-associated DNA double-strand breaks (DSBs). In this paper, we propose a new mechanism by which cells avoid Top1-induced replication-dependent DNA damage. We show that the structure-specific endonuclease Mus81-Eme1 is responsible for generating DSBs in response to Top1 inhibition and for allowing cell survival. We provide evidence that Mus81 cleaves replication forks rather than excises Top1cc’s. DNA combing demonstrated that Mus81 also allows efficient replication fork progression after CPT treatment. We propose that Mus81 cleaves stalled replication forks, which allows dissipation of the excessive supercoiling resulting from Top1 inhibition, spontaneous reversal of Top1cc, and replication fork progression.
Microarray analysis shows that transcription of 306 E. Coli genes is affected by changes in the level of chromosome supercoiling, suggesting that supercoiling transmits regulatory signals from the environment to many cellular pathways.
The chromosome of Escherichia coli is maintained in a negatively supercoiled state, and supercoiling levels are affected by growth phase and a variety of environmental stimuli. In turn, supercoiling influences local DNA structure and can affect gene expression. We used microarrays representing nearly the entire genome of Escherichia coli MG1655 to examine the dynamics of chromosome structure.
We measured the transcriptional response to a loss of supercoiling caused either by genetic impairment of a topoisomerase or addition of specific topoisomerase inhibitors during log-phase growth and identified genes whose changes are statistically significant. Transcription of 7% of the genome (306 genes) was rapidly and reproducibly affected by changes in the level of supercoiling; the expression of 106 genes increased upon chromosome relaxation and the expression of 200 decreased. These changes are most likely to be direct effects, as the kinetics of their induction or repression closely follow the kinetics of DNA relaxation in the cells. Unexpectedly, the genes induced by relaxation have a significantly enriched AT content in both upstream and coding regions.
The 306 supercoiling-sensitive genes are functionally diverse and widely dispersed throughout the chromosome. We propose that supercoiling acts as a second messenger that transmits information about the environment to many regulatory networks in the cell.
Manipulations of the DNA double helix during replication, transcription and other nucleic acid processing cause a change of DNA topology, which results in torsional stress. This stress is relaxed by DNA topoisomerases, a class of enzymes present in all domains of life. Negatively supercoiled DNA is relaxed by type IA topoisomerases that are widespread in bacteria, archaea and eukaryotes. In Escherichia coli there is conflicting data about viability of ΔtopA cells lacking topoisomerase I.
In this study we sought to clarify whether E. coli cells lacking topoisomerase I are viable by using a plasmid-based lethality assay that allowed us to investigate the phenotype of ΔtopA cells without the presence of any compensatory mutations. Our results show that cells lacking topoisomerase I show an extreme growth defect and cannot be cultured without the accumulation of compensatory mutations. This growth defect can be partially suppressed by overexpression of topoisomerase III, the other type IA topoisomerase in E. coli, suggesting that the accumulation of torsional stress is, at least partially, responsible for the lethality of ΔtopA cells. The absence of RNase HI strongly exacerbates the phenotype of cells lacking topoisomerase I, which supports the idea that the processing of RNA:DNA hybrids is vitally important in ΔtopA cells. However, we did not observe suppression of the ΔtopA phenotype by increasing the level of R-loop processing enzymes, such as RNase HI or RecG.
Our data show unambiguously that E. coli cells are not viable in the absence of DNA topoisomerase I without the presence of compensatory mutations. Furthermore, our data suggest that the accumulation of R-loops is not the primary reason for the severe growth defect of cells lacking topoisomerase I, which is in contrast to the current literature. Potential reasons for this discrepancy are discussed.
The requirements for topoisomerases in transcription of adenovirus and HeLa cell genes were analyzed using drugs that specifically inhibit either topoisomerases I or II. Cleavage of viral DNA by topoisomerases in the presence of either camptothecin or VM26 was used to determine drug concentrations that led to maximal inhibition of ligation in the cleavage and ligation step of topoisomerase I or II respectively. Inhibition of topoisomerase II with VM26 did not cause a direct reduction in transcription of adenoviral genes or HeLa cell heat shock genes. VM26 did, however, interfere with other cellular processes. It reduced nucleoside uptake into HeLa cells from the medium, and it altered the normal nuclear to cytoplasmic ratio of specific RNAs. Treatment of cells with camptothecin to inhibit topoisomerase I reduced but did not abolish transcription of viral and HeLa cell genes. Transcription mediated by both RNA polymerases I and II was reduced. Topoisomerase II did not appear to substitute for topoisomerase I in transcription since treatment of cells with VM26 and camptothecin did not reduce transcript accumulation relative to cells treated with camptothecin alone.
Cellular topoisomerases and helicases are thought to play an essential role in herpesvirus replication and gene expression and are considered to be potential targets for antiviral therapies. Topoisomerase I (Topo I) and Topo II inhibitors can selectively inhibit Epstein-Barr virus (EBV) lytic cycle DNA replication. We found that the Topo I inhibitor camptothecin and, to a lesser extent, the Topo II inhibitor etoposide are potent inhibitors of the transcription and replication function of the EBV-encoded immediate-early protein Zta (also referred to as ZEBRA, EB1, and BZLF1). Camptothecin inhibited the Zta transcription activation of endogenous and reporter-linked viral promoters. Small interfering RNA depletion of Topo I also inhibited the Zta-dependent activation of lytic cycle DNA replication. Topo I could be coimmunoprecipitated with Zta, but this interaction was restricted to EBV-positive cells, suggesting that other viral proteins stabilize the interaction between Zta and Topo I. We also found that the RecQL1 helicase, which is known to associate with Kaposi's sarcoma-associated herpesvirus (KSHV) OriLyt, interacts with EBV OriLyt. Treatment with camptothecin reduced both Zta and RecQL1 binding to OriLyt in vivo, suggesting that Topo I promotes replication protein assembly at OriLyt.
Type II DNA topoisomerases catalyse DNA double-strand cleavage, passage and re-ligation to effect topological changes. There is considerable interest in elucidating topoisomerase II roles, particularly as these proteins are targets for anti-cancer drugs. Here we uncover a role for topoisomerase IIα in RNA polymerase I-directed ribosomal RNA gene transcription, which drives cell growth and proliferation and is upregulated in cancer cells. Our data suggest that topoisomerase IIα is a component of the initiation-competent RNA polymerase Iβ complex and interacts directly with RNA polymerase I-associated transcription factor RRN3, which targets the polymerase to promoter-bound SL1 in pre-initiation complex formation. In cells, activation of rDNA transcription is reduced by inhibition or depletion of topoisomerase II, and this is accompanied by reduced transient double-strand DNA cleavage in the rDNA-promoter region and reduced pre-initiation complex formation. We propose that topoisomerase IIα functions in RNA polymerase I transcription to produce topological changes at the rDNA promoter that facilitate efficient de novo pre-initiation complex formation.
Topoisomerases facilitate the progress of elongating polymerases during transcription. Zomerdijk and colleagues now demonstrate an additional role for this enzyme; their data suggest that Top2 can cleave DNA inducing topological changes at the ribosomal DNA promoter, which assists de novo assembly of the RNA polymerase I pre-initiation complex.
DNA Topoisomerase I (Top1) is required to relax DNA supercoils generated by RNA polymerases (RNAPs). Top1 is inhibited with high specificity by camptothecin (CPT), an effective anticancer agent, and by oxidative base damage and ribonucleotides in DNA strands, resulting into Top1-DNA cleavage complexes (Top1ccs). To understand how Top1ccs affect genome stability, we have investigated the global transcriptional response to CPT-induced Top1ccs. Top1ccs trigger an accumulation of antisense RNAPII transcripts specifically at active divergent CpG-island promoters in a replication-independent and Top1-dependent manner. As CPT increases antisense transcript levels in the presence of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, a transcription inhibitor, Top1ccs likely impair antisense RNA degradation. Time-course data showed a burst of Top1ccs increased by CPT at promoter sites and along transcribed regions, causing a transient block of RNAPII at the promoter. Moreover, cell immunofluorescence analyses showed that Top1ccs induce a transient increase of R-loops specifically at highly transcribed regions such as nucleoli in a Top1-dependent manner. Thus, a specific and highly dynamic transcriptional response to Top1ccs occurs at divergent active CpG-island promoters, which may include a transient stabilization of R-loops. The results clarify molecular features of a response pathway leading to transcription-dependent genome instability and altered transcription regulation.
The role of topoisomerases in the replication of human adenovirus type 5 was investigated with topoisomerase inhibitors. Both topoisomerase I and topoisomerase II inhibitors blocked adenovirus replication when added at the time of infection. Both types of inhibitors induced strand cleavages at specific sites in the adenovirus early templates. The cleavage sites were mapped near the 5' and 3' ends of the genes transcribed early during infection. At late times after infection, camptothecin, a topoisomerase I inhibitor, inhibited adenovirus DNA replication and induced the formation of single- and double-stranded fragments with breakpoints located at defined regions of the viral genome. The topoisomerase II inhibitors, VP-16 (etoposide) and ellipticine, did not block adenovirus DNA replication and did not induce an appreciable amount of double-strand cleavages in the newly synthesized DNA. On the other hand, VP-16 promoted double-strand cleavages at specific sites of nonreplicating adenovirus DNA. The packaging of adenovirus DNA into virus particles, which contain supercoiled adenovirus DNA (M.-L. Wong and M.-T. Hsu, Nucleic Acids Res. 17:3535-3550, 1989), was inhibited by the topoisomerase II inhibitors. Transcription of adenovirus major late genes was inhibited by both topoisomerase I and topoisomerase II inhibitors. In addition, camptothecin caused a premature termination of major late transcription. Electron microscopic analysis showed that adenovirus templates late after infection were arranged in topologically constrained loop domains. Together, these data provide evidence for the requirement of topoisomerase activities in the replication, transcription, and packaging of the linear adenovirus genome.
Camptothecin stabilizes the topoisomerase I-DNA covalent intermediate that forms during the relaxation of torsionally strained DNA. By mapping the position of the resultant DNA nicks, we analyzed the distribution of the covalent intermediates formed on heat shock genes in cultured Drosophila melanogaster cells. Topoisomerase I was found to interact with the transcriptionally active genes hsp22, hsp23, hsp26, and hsp28 after heat shock but not with the inactive genes before heat shock. The interaction occurred predominantly within the transcribed region, with specific sites occurring on both the transcribed and nontranscribed strands of the DNA. Little interaction was seen with nontranscribed flanking sequences. Camptothecin only partially inhibited transcription of the hsp28 gene during heat shock, causing a reduced level of transcripts which were nonetheless full length. Topoisomerase I also interacted with the DNA throughout the transcriptionally active hsp83 gene, including an intron, in both heat-shocked and non-heat-shocked cells. The results point to a dynamic set of interactions at the active locus.
Fostriecin causes a delayed inhibition of replicative DNA synthesis in human cells, consistent with a role for DNA topoisomerase II (its target enzyme) at a late stage in replication. Fostriecin does not inhibit UV-induced excision repair. The less specific inhibitor novobiocin blocks repair in permeabilised cells given a low dose of UV, presumably through a mechanism other than the inhibition of topoisomerase II. Its effect cannot be accounted for by a depletion of the ATP required for incision. Camptothecin, an inhibitor of DNA topoisomerase I, blocks replicative DNA synthesis immediately but incompletely, suggesting a participation of topoisomerase I at the replication fork, but it, too, has no influence on DNA repair. We thus find no evidence for involvement of either topoisomerase I or II in the response of cells to UV damage.
The effects of supercoiling on the topoisomerization reaction by eukaryotic DNA topoisomerases I have been analyzed. The systems used were: DNA topoisomerase I from wheat germ, chicken erythrocyte and calf thymus on a 2.3 kb DNA fragment which encompasses the immunoglobulin kappa-light chain (L kappa) promoter of the mouse plasmacytoma MPC11; S. cerevisiae DNA topoisomerase I on a 2.2 kb DNA fragment from the same organism which encompasses the regulatory and the coding region of the ADH II gene; wheat germ DNA topoisomerase I on the plasmid pUC18. It was found in every system that lack of torsional stress prevents topoisomerization of the substrate. A simple regulatory model of DNA topoisomerase I function, based on topological considerations, is presented.
We have found that Chlamydomonas reinhardtii cells contain an ATP-dependent topoisomerase activity that supercoils circular DNA in vitro. Subsequent addition of a type I topoisomerase eliminates the supercoils. Like bacterial gyrase, this activity is inhibited by low concentrations of novobiocin (less than 0.1 microM) and by nalidixic acid (less than 0.1 microM). We have examined the effects of these topoisomerase inhibitors on accumulation of various chloroplast transcripts in vivo. Novobiocin differentially affected such transcripts; some transcripts became more abundant while many others were reduced in the presence of this drug. Nalidixic acid on the other hand caused many transcripts to become more abundant albeit to varying degrees. Inhibitors of this algal topoisomerase specifically stimulate a family of related transcripts which we have previously shown to be under light-dark control. We discuss how the inhibitors of this topoisomerase might exert their in vivo effects.
The azafluoranthene alkaloid eupolauridine has previously been shown to have in vitro antifungal activity and selective inhibition of fungal topoisomerase I. The present study was undertaken to examine further its selectivity and mode of action. Eupolauridine completely inhibits the DNA relaxation activity of purified fungal topoisomerase I at 50 μg/ml, but it does not stabilize the cleavage complex of either human or fungal topoisomerase I. Cleavage complex stabilization is the mode of action of topoisomerase I targeting drugs of the camptothecin family. Also, unlike camptothecin, eupolauridine does not cause significant cytotoxicity in mammalian cells. To determine if the inhibition of topoisomerase I is the principal mode of antifungal action of eupolauridine, Saccharomyces cerevisiae strains with alterations in topoisomerase genes were used in clonogenic assays. The antifungal activity of eupolauridine was not diminished in the absence of topoisomerase I; rather, the cells lacking the enzyme were more sensitive to the drug. Cell-killing activity of eupolauridine was also more pronounced in cells that overexpressed topoisomerase II. In vitro assays with the purified yeast enzyme confirmed that eupolauridine stabilized topoisomerase II covalent complexes. These results indicate that a major target for fungal cell killing by eupolauridine is DNA topoisomerase II rather than topoisomerase I, but does not exclude the possibility that the drug also acts against other targets.
Inactivation of the nonessential TOP1 gene, which codes for Saccharomyces cerevisiae DNA topoisomerase I, affects the rate of transcription starting at the ADH2 promoter. For both the chromosomal gene and the plasmid-borne promoter, mRNA accumulation is kinetically favored in the mutant relative to a wild-type isogenic strain. The addition of ethanol causes in wild-type yeast strains a substantial increase in linking number both on the ADH2-containing plasmid and on the resident 2 microns DNA. Evidence has been obtained that such an in vivo increase in linking number depends on (i) the activity of DNA topoisomerase I and of no other enzyme and (ii) ethanol addition, not on the release from glucose repression. A direct cause-effect relationship between the change in supercoiling and alteration of transcription cannot be defined. However, the hypothesis that a metabolism-induced modification of DNA topology in a eukaryotic cell plays a role in regulating gene expression is discussed.
The potent novel poly(ADP-ribose) polymerase (PARP) inhibitor, NU1025, enhances the cytotoxicity of DNA-methylating agents and ionizing radiation by inhibiting DNA repair. We report here an investigation of the role of PARP in the cellular responses to inhibitors of topoisomerase I and II using NU1025. The cytotoxicity of the topoisomerase I inhibitor, camptothecin, was increased 2.6-fold in L1210 cells by co-incubation with NU1025. Camptothecin-induced DNA strand breaks were also increased 2.5-fold by NU1025 and exposure to camptothecin-activated PARP. In contrast, NU1025 did not increase the DNA strand breakage or cytotoxicity caused by the topoisomerase II inhibitor etoposide. Exposure to etoposide did not activate PARP even at concentrations that caused significant levels of apoptosis. Taken together, these data suggest that potentiation of camptothecin cytotoxicity by NU1025 is a direct result of increased DNA strand breakage, and that activation of PARP by camptothecin-induced DNA damage contributes to its repair and consequently cell survival. However, in L1210 cells at least, it would appear that PARP is not involved in the cellular response to etoposide-mediated DNA damage. On the basis of these data, PARP inhibitors may be potentially useful in combination with topoisomerase I inhibitor anticancer chemotherapy. © 2001 Cancer Research Campaign http://www.bjcancer.com
poly(ADP-ribose) polymerase; topoisomerase I; topoisomerase II; cytotoxicity; DNA damage
An inhibitor of RNA polymerase II transcription in vitro has been purified from HeLa cell nuclear extracts. Partial amino acid sequences derived from the purified protein revealed that the inhibitor of transcription corresponded to human topoisomerase II. Order of addition experiments provided evidence indicating that topoisomerase II inhibited transcription by binding over the core promoter and blocking preinitiation complex formation. Topoisomerase II-mediated repression could be relieved by sequence-specific transcriptional activators, having different activating and/or DNA binding domains, but antirepression required a transcriptional activation function in addition to a DNA binding domain. Moreover, transcription by RNA polymerase I was also inhibited by topoisomerase II and this inhibition could be relieved by the RNA polymerase I transactivator UBF. These observations suggest that topoisomerase II may participate in a general repression of transcription which can be counteracted by transcriptional activators.
The superhelicity of the chromosome, which is controlled by DNA topoisomerases, modulates global gene expression. Investigations of transcriptional responses to the modulation of gyrase function have identified two types of topoisomerase-mediated transcriptional responses: (i) steady-state changes elicited by a mutation in gyrase, such as the D82G mutation in GyrA, and (ii) dynamic changes elicited by the inhibition of gyrase. We hypothesize that the steady-state effects are due to the changes in biochemical properties of gyrase, whereas the dynamic effects are due to an imbalance between supercoiling and relaxation activities, which appears to be influenced by the RecA activity. Herein, we present biochemical evidence for hypothesized mechanisms. GyrA D82G gyrase exhibits a reduced supercoiling activity. The RecA protein can influence the balance between supercoiling and relaxation activities either by interfering with the activity of DNA gyrase or by facilitating the relaxation reaction. RecA has no effect on the supercoiling activity of gyrase but stimulates the relaxation activity of topoisomerase I. This stimulation is specific and requires formation of an active RecA filament. These results suggest that the functional interaction between RecA and topoisomerase I is responsible for RecA-mediated modulation of the relaxation-dependent transcriptional activity of the Escherichia coli chromosome.
The anti-cancer drug camptothecin inhibits replication and transcription by trapping DNA topoisomerase I (Top1) covalently to DNA in a “cleavable complex”. To examine the effects of camptothecin on RNA synthesis genome-wide we used Bru-Seq and show that camptothecin treatment primarily affected transcription elongation. We also observed that camptothecin increased RNA reads past transcription termination sites as well as at enhancer elements. Following removal of camptothecin, transcription spread as a wave from the 5’-end of genes with no recovery of transcription apparent from RNA polymerases stalled in the body of genes. As a result, camptothecin preferentially inhibited the expression of large genes such as proto-oncogenes, and anti-apoptotic genes while smaller ribosomal protein genes, pro-apoptotic genes and p53 target genes showed relative higher expression. Cockayne syndrome group B fibroblasts (CS-B), which are defective in transcription-coupled repair (TCR), showed an RNA synthesis recovery profile similar to normal fibroblasts suggesting that TCR is not involved in the repair of or RNA synthesis recovery from transcription-blocking Top1 lesions. These findings of the effects of camptothecin on transcription have important implications for its anti-cancer activities and may aid in the design of improved combinatorial treatments involving Top1 poisons.
Human topoisomerase I catalyzes the relaxation of DNA supercoils in fundamental cell processes like transcription, replication and chromosomal segregation. It is the only target of the camptothecin family of anticancer drugs. Among these, topotecan has been used to treat lung and ovarian carcinoma for several years. Camptothecins reversibly binds to the covalent intermediate DNA-enzyme, stabilizing the cleavable complex and reducing the religation rate. The stalled complex then collides with the progression of the replication fork, producing lethal double strand DNA breaks and eventually cell death.
Long lasting molecular dynamics simulations of the DNA-topoisomerase I binary complex and of the DNA-topoisomerase-topotecan ternary complex have been performed and compared. The conformational space sampled by the binary complex is reduced by the presence of the drug, as observed by principal component and cluster analyses. This conformational restraint is mainly due to the reduced flexibility of residues 633–643 (the region connecting the linker to the core domain) that causes an overall mobility loss in the ternary complex linker domain. During the simulation, DNA/drug stacking interactions are fully maintained, and hydrogen bonds are maintained with the enzyme. Topotecan keeps the catalytic residue Lys532 far from the DNA, making it unable to participate to the religation reaction. Arg364 is observed to interact with both the B and E rings of topotecan with two stable direct hydrogen bonds. An interesting constrain exerted by the protein on the geometrical arrangement of topotecan is also observed.
Atomistic-scale understanding of topotecan interactions with the DNA-enzyme complex is fundamental to the explaining of its poisonous effect and of the drug resistance observed in several single residue topoisomerase mutants. We observed significant alterations due to topotecan in both short-range interactions and long-range protein domain communications.
Fluoroquinolones are broad-spectrum antimicrobial agents that target type II topoisomerases. Many fluoroquinolones are highly specific for bacterial type II topoisomerases and act against both DNA gyrase and topoisomerase IV. In Escherichia coli, mutations causing quinolone resistance are often found in the gene that encodes the A subunit of DNA gyrase. One common site for resistance-conferring mutations alters Ser83, and mutations to Leu or Trp result in high levels of resistance to fluoroquinolones. In the present study we demonstrate that the mutation of Ser83 to Trp in DNA gyrase (GyrS83W) also results in sensitivity to agents that are potent inhibitors of eukaryotic topoisomerase II but that are normally inactive against prokaryotic enzymes. Epipodophyllotoxins, such as etoposide, teniposide and amino-azatoxin, inhibited the DNA supercoiling activity of GyrS83W, and the enzyme caused elevated levels of DNA cleavage in the presence of these agents. The DNA sequence preference for GyrS83W-induced cleavage sites in the presence of etoposide was similar to that seen with eukaryotic type II topoisomerases. Introduction of the GyrS83W mutation in E. coli strain RFM443-242 by site-directed mutagenesis sensitized it to epipodophyllotoxins and amino-azatoxin. Our results demonstrate that sensitivity to agents that target topoisomerase II is conserved between prokaryotic and eukaryotic enzymes, suggesting that drug interaction domains are also well conserved and likely occur in domains important for the biochemical activities of the enzymes.
Topoisomerases I and II (topo I and topo II) are nuclear enzymes functioning to resolve DNA topological problems during replication, transcription, and other DNA processes. We tested the effects of camptothecin and VP16, specific inhibitors of topo I and II, respectively, on the DNA replication of parvoviruses LuIII and H-1 and found that viral DNA synthesis was suppressed by camptothecin but not by VP16. Transcription of H-1 virus was measured by a nuclear runoff assay and showed no inhibition by camptothecin. Interestingly, topo I in the LuIII virus-infected cell nuclear extract appears to have more activity for covalently binding to viral DNA than that in mock-infected cell nuclear extracts. Our data suggested that this activity was not due to an increased transcription of the topo I gene or to greater amounts of topo I.
Camptothecin is a plant alkaloid that specifically binds topoisomerase I, inhibiting its activity and inducing double stranded breaks in DNA, activating the cell responses to DNA damage and, in response to severe treatments, triggering cell death.
Comparative transcriptomic and proteomic analyses of maize embryos that had been exposed to camptothecin were conducted. Under the conditions used in this study, camptothecin did not induce extensive degradation in the genomic DNA but induced the transcription of genes involved in DNA repair and repressed genes involved in cell division. Camptothecin also affected the accumulation of several proteins involved in the stress response and induced the activity of certain calcium-dependent nucleases. We also detected changes in the expression and accumulation of different genes and proteins involved in post-translational regulatory processes.
This study identified several genes and proteins that participate in DNA damage responses in plants. Some of them may be involved in general responses to stress, but others are candidate genes for specific involvement in DNA repair. Our results open a number of new avenues for researching and improving plant resistance to DNA injury.