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.     

Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I

Camptothecin, a cytotoxic drug, is a strong inhibitor of nucleic acid synthesis in mammalian cells and a potent inducer of strand breaks in chromosomal DNA. Neither the equilibrium dialysis nor the unwinding measurement indicates any interaction between camptothecin and purified DNA. However, camptothecin induces extensive single strand DNA breaks in reactions containing purified mammalian DNA topoisomerase I. DNA breakage in vitro is immediate and reversible. Analyses of camptothecin-induced DNA breaks show that topoisomerase I is covalently linked to the 3' end of the broken DNA. In addition, camptothecin inhibits the catalytic activity of mammalian DNA topoisomerase I. We propose that camptothecin blocks the rejoining step of the breakage-reunion reaction of mammalian DNA topoisomerase I. This blockage results in the accumulation of a cleavable complex which resembles the transient intermediate proposed for eukaryotic DNA topoisomerase I. The inhibition of nucleic acid synthesis and the induction of DNA strand breaks observed in vivo may be related to the formation of this drug-induced cleavable complex.     

Telomeric DNA damage by topoisomerase I. A possible mechanism for cell killing by camptothecin

Topoisomerase I adjusts torsional stress in the genome by breaking and resealing one strand of the helix through a transient covalent coupling between enzyme and DNA. Camptothecin, a specific topoisomerase I poison, traps this covalent intermediate, thereby damaging the genome. Here we examined the activity of topoisomerase I at telomeric repeats to determine whether telomere structures are targets for DNA damage. We show that topoisomerase I is catalytically active in cleaving the G-rich telomeric strand in vitro in the presence of camptothecin but not in cleaving the C-rich strand. The topoisomerase I cleavage site is 5'-TT (downward arrow) AGGG-3' (cleavage site marked by the downward arrow). We also show that endogenous topoisomerase I can access telomeric DNA in vivo and form camptothecin-dependent covalent complexes. Therefore, each telomeric repeat represents a potential topoisomerase I cleavage site in vivo. Because telomere structures are comprised of a large number of repeats, telomeres in fact represent a high concentration of nested topoisomerase I sites. Therefore, more telomeric DNA damage by camptothecin could occur in cells with longer telomeres when cells possess equivalent levels of topoisomerase I. The evidence presented here suggests that DNA damage at telomeric repeats by topoisomerase I is a prominent feature of cell killing by camptothecin and triggers camptothecin-induced apoptosis.     

Mapping Topoisomerase Sites in Mitochondrial DNA with a Poisonous Mitochondrial Topoisomerase I (Top1mt)

Mitochondrial topoisomerase I (Top1mt) is a type IB topoisomerase present in vertebrates and exclusively targeted to mitochondria. Top1mt relaxes mitochondrial DNA (mtDNA) supercoiling by introducing transient cleavage complexes wherein the broken DNA strand swivels around the intact strand. Top1mt cleavage complexes (Top1mtcc) can be stabilized in vitro by camptothecin (CPT). However, CPT does not trap Top1mtcc efficiently in cells and is highly cytotoxic due to nuclear Top1 targeting. To map Top1mtcc on mtDNA in vivo and to overcome the limitations of CPT, we designed two substitutions (T546A and N550H) in Top1mt to stabilize Top1mtcc. We refer to the double-mutant enzyme as Top1mt*. Using retroviral transduction and ChIP-on-chip assays with Top1mt* in Top1mt knock-out murine embryonic fibroblasts, we demonstrate that Top1mt* forms high levels of cleavage complexes preferentially in the noncoding regulatory region of mtDNA, accumulating especially at the heavy strand replication origin O_H, in the ribosomal genes (12S and 16S) and at the light strand replication origin O_L. Expression of Top1mt* also caused rapid mtDNA depletion without affecting mitochondria mass, suggesting the existence of specific mitochondrial pathways for the removal of damaged mtDNA.     

The intra-S-phase checkpoint affects both DNA replication initiation and elongation: single-cell and -DNA fiber analyses

To investigate the contribution of DNA replication initiation and elongation to the intra-S-phase checkpoint, we examined cells treated with the specific topoisomerase I inhibitor camptothecin. Camptothecin is a potent anticancer agent producing well-characterized replication-mediated DNA double-strand breaks through the collision of replication forks with topoisomerase I cleavage complexes. After a short dose of camptothecin in human colon carcinoma HT29 cells, DNA replication was inhibited rapidly and did not recover for several hours following drug removal. That inhibition occurred preferentially in late-S-phase, compared to early-S-phase, cells and was due to both an inhibition of initiation and elongation, as determined by pulse-labeling nucleotide incorporation in replication foci and DNA fibers. DNA replication was actively inhibited by checkpoint activation since 7-hydroxystaurosporine (UCN-01), the specific Chk1 inhibitor CHIR-124, or transfection with small interfering RNA targeting Chk1 restored both initiation and elongation. Abrogation of the checkpoint markedly enhanced camptothecin-induced DNA damage at replication sites where histone γ-H2AX colocalized with replication foci. Together, our study demonstrates that the intra-S-phase checkpoint is exerted by Chk1 not only upon replication initiation but also upon DNA elongation.     

WRN protects against topo I but not topo II inhibitors by preventing DNA break formation

The Werner syndrome helicase/3′-exonuclease (WRN) is a major component of the DNA repair and replication machinery. To analyze whether WRN is involved in the repair of topoisomerase-induced DNA damage we utilized U2-OS cells, in which WRN is stably down-regulated (wrn-kd), and the corresponding wild-type cells (wrn-wt). We show that cells not expressing WRN are hypersensitive to the toxic effect of the topoisomerase I inhibitor topotecan, but not to the topoisomerase II inhibitor etoposide. This was shown by mass survival assays, colony formation and induction of apoptosis. Upon topotecan treatment WRN deficient cells showed enhanced DNA replication inhibition and S-phase arrest, whereas after treatment with etoposide they showed the same cell cycle response as the wild-type. A considerable difference between WRN and wild-type cells was observed for DNA single- and double-strand break formation in response to topotecan. Topotecan induced DNA single-strand breaks 6 h after treatment. In both wrn-wt and wrn-kd cells these breaks were repaired at similar kinetics. However, in wrn-kd but not wrn-wt cells they were converted into DNA double-strand breaks (DSBs) at high frequency, as shown by neutral comet assay and phosphorylation of H2AX. Our data provide evidence that WRN is involved in the repair of topoisomerase I, but not topoisomerase II-induced DNA damage, most likely via preventing the conversion of DNA single-strand breaks into DSBs during the resolution of stalled replication forks at topo I–DNA complexes. We suggest that the WRN status of tumor cells impacts anticancer therapy with topoisomerase I, but not topoisomerase II inhibitors.     

Human REV3 DNA Polymerase Zeta Localizes to Mitochondria and Protects the Mitochondrial Genome

To date, mitochondrial DNA polymerase γ (POLG) is the only polymerase known to be present in mammalian mitochondria. A dogma in the mitochondria field is that there is no other polymerase present in the mitochondria of mammalian cells. Here we demonstrate localization of REV3 DNA polymerase in the mammalian mitochondria. We demonstrate localization of REV3 in the mitochondria of mammalian tissue as well as cell lines. REV3 associates with POLG and mitochondrial DNA and protects the mitochondrial genome from DNA damage. Inactivation of Rev3 leads to reduced mitochondrial membrane potential, reduced OXPHOS activity, and increased glucose consumption. Conversely, inhibition of the OXPHOS increases expression of Rev3. Rev3 expression is increased in human primary breast tumors and breast cancer cell lines. Inactivation of Rev3 decreases cell migration and invasion, and localization of Rev3 in mitochondria increases survival and the invasive potential of cancer cells. Taken together, we demonstrate that REV3 functions in mammalian mitochondria and that mitochondrial REV3 is associated with the tumorigenic potential of cells.     

PARP-1 ensures regulation of replication fork progression by homologous recombination on damaged DNA

Poly-ADP ribose polymerase 1 (PARP-1) is activated by DNA damage and has been implicated in the repair of single-strand breaks (SSBs). Involvement of PARP-1 in other DNA damage responses remains controversial. In this study, we show that PARP-1 is required for replication fork slowing on damaged DNA. Fork progression in PARP-1^−/− DT40 cells is not slowed down even in the presence of DNA damage induced by the topoisomerase I inhibitor camptothecin (CPT). Mammalian cells treated with a PARP inhibitor or PARP-1–specific small interfering RNAs show similar results. The expression of human PARP-1 restores fork slowing in PARP-1^−/− DT40 cells. PARP-1 affects SSB repair, homologous recombination (HR), and nonhomologous end joining; therefore, we analyzed the effect of CPT on DT40 clones deficient in these pathways. We find that fork slowing is correlated with the proficiency of HR-mediated repair. Our data support the presence of a novel checkpoint pathway in which the initiation of HR but not DNA damage delays the fork progression.     

CFS-1686 Causes Cell Cycle Arrest at Intra-S Phase by Interference of Interaction of Topoisomerase 1 with DNA

CFS-1686 (chemical name (E)-N-(2-(diethylamino)ethyl)-4-(2-(2-(5-nitrofuran-2-yl)vinyl)quinolin-4-ylamino)benzamide) inhibits cell proliferation and triggers late apoptosis in prostate cancer cell lines. Comparing the effect of CFS-1686 on cell cycle progression with the topoisomerase 1 inhibitor camptothecin revealed that CFS-1686 and camptothecin reduced DNA synthesis in S-phase, resulting in cell cycle arrest at the intra-S phase and G1-S boundary, respectively. The DNA damage in CFS-1686 and camptothecin treated cells was evaluated by the level of ATM phosphorylation, γH2AX, and γH2AX foci, showing that camptothecin was more effective than CFS-1686. However, despite its lower DNA damage capacity, CFS-1686 demonstrated 4-fold higher inhibition of topoisomerase 1 than camptothecin in a DNA relaxation assay. Unlike camptothecin, CFS-1686 demonstrated no activity on topoisomerase 1 in a DNA cleavage assay, but nevertheless it reduced the camptothecin-induced DNA cleavage of topoisomerase 1 in a dose-dependent manner. Our results indicate that CFS-1686 might bind to topoisomerase 1 to inhibit this enzyme from interacting with DNA relaxation activity, unlike campothecin''s induction of a topoisomerase 1-DNA cleavage complex. Finally, we used a computer docking strategy to localize the potential binding site of CFS-1686 to topoisomerase 1, further indicating that CFS-1686 might inhibit the binding of Top1 to DNA.     

Inactivation of Saccharomyces cerevisiae OGG1 DNA repair gene leads to an increased frequency of mitochondrial mutants

The OGG1 gene encodes a highly conserved DNA glycosylase that repairs oxidized guanines in DNA. We have investigated the in vivo function of the Ogg1 protein in yeast mitochondria. We demonstrate that inactivation of ogg1 leads to at least a 2-fold increase in production of spontaneous mitochondrial mutants compared with wild-type. Using green fluorescent protein (GFP) we show that a GFP–Ogg1 fusion protein is transported to mitochondria. However, deletion of the first 11 amino acids from the N-terminus abolishes the transport of the GFP–Ogg1 fusion protein into the mitochondria. This analysis indicates that the N-terminus of Ogg1 contains the mitochondrial localization signal. We provide evidence that both yeast and human Ogg1 proteins protect the mitochondrial genome from spontaneous, as well as induced, oxidative damage. Genetic analyses revealed that the combined inactivation of OGG1 and OGG2 [encoding an isoform of the Ogg1 protein, also known as endonuclease three-like glycosylase I (Ntg1)] leads to suppression of spontaneously arising mutations in the mitochondrial genome when compared with the ogg1 single mutant or the wild-type. Together, these studies provide in vivo evidence for the repair of oxidative lesions in the mitochondrial genome by human and yeast Ogg1 proteins. Our study also identifies Ogg2 as a suppressor of oxidative mutagenesis in mitochondria.     

POS5 gene of Saccharomyces cerevisiae encodes a mitochondrial NADH kinase required for stability of mitochondrial DNA

In a search for nuclear genes that affect mutagenesis of mitochondrial DNA in Saccharomyces cerevisiae, an ATP-NAD (NADH) kinase, encoded by POS5, that functions exclusively in mitochondria was identified. The POS5 gene product was overproduced in Escherichia coli and purified without a mitochondrial targeting sequence. A direct biochemical assay demonstrated that the POS5 gene product utilizes ATP to phosphorylate both NADH and NAD⁺, with a twofold preference for NADH. Disruption of POS5 increased minus-one frameshift mutations in mitochondrial DNA 50-fold, as measured by the arg8^m reversion assay, with no increase in nuclear mutations. Also, a dramatic increase in petite colony formation and slow growth on glycerol or limited glucose were observed. POS5 was previously described as a gene required for resistance to hydrogen peroxide. Consistent with a role in the mitochondrial response to oxidative stress, a pos5 deletion exhibited a 28-fold increase in oxidative damage to mitochondrial proteins and hypersensitivity to exogenous copper. Furthermore, disruption of POS5 induced mitochondrial biogenesis as a response to mitochondrial dysfunction. Thus, the POS5 NADH kinase is required for mitochondrial DNA stability with a critical role in detoxification of reactive oxygen species. These results predict a role for NADH kinase in human mitochondrial diseases.     

Etoposide and Camptothecin Reduce Growth,Viability, the Generation of Petite Mutants,and Recognize the Active Site of DNA Topoisomerase I and II Enzymes in Candida glabrata

Candidemia, one of the most common invasive fungal infections in hospitalized patients, can lead to death and huge financial losses. Candida albicans is the main causative agent of this disorder and Candida glabrata occupies the second or third place, for which new therapeutic alternatives must be found. The objective of the present study was to evaluate the inhibitory effect of etoposide and camptothecin (inhibitors of deoxyribonucleic acid (DNA) topoisomerase) on the C. glabrata CBS138 strain. Etoposide and camptothecin showed better or similar MIC (minimum inhibitory concentration) (5 and 2.5 μg/mL, respectively), with respect to fluconazole (8 μg/mL) and itraconazole (4 μg/mL). They also suppressed colony formation during the 12-h test. On the other hand, petite colonies were less formed by exposing C. glabrata to etoposide or camptothecin (indicating low toxicity), with respect fluconazole and itraconazole. Such colonies are phenotypically observed as limited growth in medium containing a non-fermentable carbon source, and are genotypically characterized by a partial or total loss of mitochondrial DNA (mtDNA) fragments. Using PCR techniques and cell staining with 4′,6-diamidino-2-phenylindole (DAPI), loss of mtDNA was detected only in yeast cells treated with fluconazole. Additionally, molecular docking studies with etoposide and camptothecin showed recognition in the active site of the Topo I and II enzymes from C. glabrata. Since etoposide and camptothecin showed good inhibitory activity and low toxicity on C. glabrata; they should certainly be of interest for the treatment of C. glabrata infections and the design and development of new antifungal compounds derived from these drugs.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12088-021-00942-6.     

Spatial organization of topoisomerase I-mediated DNA cleavage induced by camptothecin-oligonucleotide conjugates

Triple helix-forming oligonucleotides covalently linked to topoisomerase I inhibitors, in particular the antitumor agent camptothecin, trigger topoisomerase I-mediated DNA cleavage selectively in the proximity of the binding site of the oligonucleotide vector. In the present study, we have performed a systematic analysis of the DNA cleavage efficiency as a function of the positioning of the camptothecin derivative, either on the 3′ or the 5′ side of the triplex, and the location of the cleavage site. A previously identified cleavage site was inserted at different positions within two triplex site-containing 59 bp duplexes. Sequence-specific DNA cleavage by topoisomerase I occurs only with triplex conjugates bearing the inhibitor at the 3′-end of the oligonucleotide and on the oligopyrimidine strand of the duplex. The lack of targeted cleavage on the 5′ side is attributed to the structural differences of the 3′ and 5′ duplex–triplex DNA junctions. The changes induced in the double helix by the triple-helical structure interfere with the action of the enzyme according to a preferred spatial organization. Camptothecin conjugates of oligonucleotides provide efficient tools to probe the organization of the topoisomerase I–DNA complex and will be useful to understand the functioning of topoisomerase I in living cells.     

SUMO-1 conjugation to human DNA topoisomerase II isozymes

Topoisomerase I-mediated DNA damage induced by camptothecin has been shown to induce rapid small ubiquitin-related modifier (SUMO)-1 conjugation to topoisomerase I. In the current study, we show that topoisomerase II-mediated DNA damage induced by teniposide (VM-26) results in the formation of high molecular weight conjugates of both topoisomerase IIalpha and IIbeta isozymes in HeLa cells. Immunological characterization of these conjugates suggests that both topoisomerase IIalpha and IIbeta isozymes are conjugated to SUMO-1. The involvement of SUMO-1/UBC9 in the modification of topoisomerase II isozymes is also supported by the demonstration of physical interaction between topoisomerase II and SUMO-1/UBC9. Surprisingly, ICRF-193, which does not induce topoisomerase II-mediated DNA damage but traps topoisomerase II into a circular clamp conformation, is also shown to induce similar SUMO-1 conjugation to topoisomerase II isozymes. In addition, we show that both oxidative and heat shock stresses, which can cause protein damage, rapidly increase nuclear SUMO-1 conjugates. These studies raise the question on whether SUMO-1 conjugation to topoisomerases is an indirect result of a DNA damage response or a direct result because of protein conformational changes.     

Comparison of responses of DNA topoisomerase I from Candida albicans and human cells to four new agents which stimulate topoisomerase-dependent DNA nicking

Abstract DNA topoisomerase I is a potential target for therapeutic antifungal agents predicted to have a fungicidal mode of action. This report describes four agents with varying degrees of selectivity for the fungal topoisomerase I compared to the human enzyme: 5-hydroxy-1H-indole-3-acetic acid (5-HIAA), quinizarin, dibenzo- p -dioxin-2-carboxylic acid and 7-amino-4-hydroxy-2-naphthalenesulfonic acid. Taken together with the response of topoisomerase to camptothecin and aminocatechol, these data suggest that there are sufficient structural differences between the topoisomerase I from Candida albicans and human cells to allow selective targeting of the fungal topoisomerase I over its human counterpart.     

The Schizosaccharomyces pombe Pfh1p DNA helicase is essential for the maintenance of nuclear and mitochondrial DNA

Schizosaccharomyces pombe Pfh1p is an essential member of the Pif family of 5′-3′ DNA helicases. The two Saccharomyces cerevisiae homologs, Pif1p and Rrm3p, function in nuclear DNA replication, telomere length regulation, and mitochondrial genome integrity. We demonstrate here the existence of multiple Pfh1p isoforms that localized to either nuclei or mitochondria. The catalytic activity of Pfh1p was essential in both cellular compartments. The absence of nuclear Pfh1p resulted in G₂ arrest and accumulation of DNA damage foci, a finding suggestive of an essential role in DNA replication. Exogenous DNA damage resulted in localization of Pfh1p to DNA damage foci, suggesting that nuclear Pfh1p also functions in DNA repair. The absence of mitochondrial Pfh1p caused rapid depletion of mitochondrial DNA. Despite localization to nuclei and mitochondria in S. pombe, neither of the S. cerevisiae homologs, nor human PIF1, suppressed the lethality of pfh1Δ cells. However, the essential nuclear function of Pfh1p could be supplied by Rrm3p. Expression of Rrm3p suppressed the accumulation of DNA damage foci but not the hydroxyurea sensitivity of cells depleted of nuclear Pfh1p. Together, these data demonstrate that Pfh1p has essential roles in the replication of both nuclear and mitochondrial DNA.     

Characterization of a camptothecin-resistant human DNA topoisomerase I in an in vitro system for Simian virus 40 DNA replication.

DNA topoisomerase I was required for bidirectional DNA replication in an in vitro system for Simian virus 40 (SV40) DNA replication with purified proteins in which the replication fork moved at the rate of 260 nucleotides/min on average. DNA topoisomerase I purified from camptothecin-resistant human lymphoblastoid cells, which confers high resistance of cellular DNA replication to camptothecin [Andoh, T., Ishii, K., Suzuki, Y., Ikegami, Y., Kusunoki, Y., Takemoto, Y. & Okada, K. (1987) Proc. Natl Acad. Sci. USA 84, 5565-5569], was characterized using this system. The activity of stimulating bidirectional DNA replication was comparable between two topoisomerase I from parental and resistant cells, i.e. in its dose-response relationship and in its time course for DNA synthesis. Camptothecin severely inhibited the leading as well as the lagging strand synthesis in the reaction containing the wild type topoisomerase I but not the mutant type topoisomerase I. The mutant type topoisomerase I was over 125-fold as resistant to camptothecin as the wild type topoisomerase I. These results are in good agreement with those on the sensitivity of cellular DNA synthesis to camptothecin in the resistant cells. These findings suggest that topoisomerase I is involved in cellular DNA replication as a swivelase and the mutation conferring camptothecin-resistance on the enzyme does not affect its functional efficiency in this system.     

Mitochondrial DNA topoisomerase I from human platelets.

An anucleated cell system has been used for the first time to study mitochondrial topoisomerase activity. Mitochondrial extracts from human blood platelets contained type I topoisomerase. The type I classification was based on ATP-independent activity, inhibition by ATP or camptothecin, and the lack of inhibition by novobiocin. Platelet mitochondrial topoisomerase I relaxation activity was inhibited linearly by increasing concentrations of EGTA. Topoisomerase activity greater than 90% inhibited by 175 microM EGTA was partially restored to 16 and 50% of the initial level of activity by the subsequent addition of 50 and 100 microM Ca2+, respectively. Additionally, results from studies of partially purified platelet mitochondrial topoisomerase I were consistent with the crude extract data. This work supports the hypothesis that platelet mitochondria contain a type I topoisomerase that is biochemically distinct from that previously isolated and characterized from cell nuclei.