Plant DMA topoisomerase I is recognized and inhibited by human Scl-70 sera autoantibodies

Type I topoisomerases (EC 5.99.1.2) are those enzymes capable of relaxing negatively supercoiled DNA without the need for ATP. The central role played by these enzymes in cell function suggests that the structure of type I topoisomerases may be highly conserved in eukaryotic cells. However, the extent of the conservation among eukaryotes is unknown. Human DNA topoisomerase I is an autoimmune antigen (Scl-70) of scleroderma patients. We have found that the autoimmune antibodies in human Scl-70 sera recognize protein from various plants, and these proteins display DNA relaxation function. In addition, Scl-70 antibodies were able to inhibit enzymatic activity of plant topoisomerase I. Therefore, the immunological cross-reactivity of the plant topoisomerase with human antibodies demonstrates that, despite divergence of eukaryotic organisms, these plant and animal enzymes retain structurally similar enzymatic features.     

Plant DNA topoisomerase I is recognized and inhibited by human Scl-70 sera autoantibodies

Type I topoisomerases (EC 5.99.1.2) are those enzymes capable of relaxing negatively supercoiled DNA without the need for ATP. The central role played by these enzymes in cell function suggests that the structure of type I topoisomerases may be highly conserved in eukaryotic cells. However, the extent of the conservation among eukaryotes is unknown. Human DNA topoisomerase I is an autoimmune antigen (Scl-70) of scleroderma patients. We have found that the autoimmune antibodies in human Scl-70 sera recognize protein from various plants, and these proteins display DNA relaxation function. In addition, Scl-70 antibodies were able to inhibit enzymatic activity of plant topoisomerase I. Therefore, the immunological cross-reactivity of the plant topoisomerase with human antibodies demonstrates that, despite divergence of eukaryotic organisms, these plant and animal enzymes retain structurally similar enzymatic features.     

Sequence-dependent effect of camptothecin on human topoisomerase I DNA cleavage

We have studied the effect of the antitumor drug, camptothecin, on the interaction of human topoisomerase I with DNA at the sequence level. At a low molar ratio of enzyme to DNA, cleavage is prominent and unique, located at a previously described hexadecameric recognition sequence, while a number of strong additional cleavage sites appear in the presence of the drug. Camptothecin stimulates cleavage at the recognition sequence less than twofold, whereas cleavage at the additional sites is stimulated up to 200-fold. Camptothecin greatly enhances the stability of the cleavable complexes formed at the additional sites, whereas the complex formed at the hexadecameric sequence is only marginally affected. Cleavage was eliminated at certain sites in the presence of camptothecin. Taken together these observations demonstrate that at least three types of potential eukaryotic topoisomerase I cleavage sites can be distinguished by the use of camptothecin. Comparison of the sequences at the additional cleavage sites in the presence of camptothecin reveals that the most frequently cleaved dinucleotide is TG with no consensus for the flanking nucleotides.     

Human topoisomerase I cleavage complexes are repaired by a p53-stimulated recombination-like reaction in vitro

Several studies have shown that human topoisomerase I (htopoI) cleaves in the vicinity of various DNA lesions and thereby forms covalent intermediates known as ‘cleavage complexes’. Such complexes are detrimental to cells if they are not repaired. Therefore, it is generally accepted that repair pathways must exist for such lesions. We have demonstrated that a htopoI cleavage complex can be recognized by a second topoisomerase I molecule and thereby perform a so-called htopoI ‘double cleavage’ in vitro. In addition, we found that the double cleavage reaction was stimulated by p53. Here we show that the double cleavage reaction results in the removal of the original htopoI cleavage complex and the generation of a single-stranded gap of ~13 nt. This gap supports a sequence-dependent DNA recombination reaction mediated by the second htopoI molecule. Furthermore, we show that p53 strongly stimulates the recombination reaction. We suggest that this reaction may represent a novel p53-dependent topoisomerase I-induced recombination repair (TIRR) pathway for htopoI cleavage complexes.     

SF2/ASF protein inhibits camptothecin-induced DNA cleavage by human topoisomerase I.

A splicing factor SF2/ASF is a natural substrate for the kinase activity of human topoisomerase I. This study demonstrates that SF2/ASF inhibits DNA cleavage by human topoisomerase I induced by the anti-cancer agent camptothecin. The inhibition is independent of the phosphorylation status of SF2/ASF. We show that the inhibition did not result from binding of SF2/ASF to DNA that would hinder interactions between topoisomerase I and DNA. Neither it was a consequence of a loss of sensitivity of the enzyme to camptothecin. We provide evidence pointing to reduced formation of the cleavable complex in the presence of SF2/ASF as a primary reason for the inhibition. This effect of SF2/ASF is reflected by inhibition of DNA relaxation catalysed by topoisomerase I.     

Identification of specific nonbridging phosphate oxygens important for DNA cleavage by human topoisomerase I

Methylphosphonate-bearing oligonucleotides are characterized by the replacement of one of the nonbridging oxygen atoms with a methyl group. While neutralizing the negative charge associated with the phosphodiester at the point of substitution, the methyl group also imparts chirality to the phosphorus atom. Herein we report the synthesis of a number of oligonucleotides containing isomerically pure S(p) and R(p) methylphosphonates at single positions for the purpose of investigating the hydrogen-bonding contacts necessary for human topoisomerase I function. It was possible to correlate these data to the recent X-ray crystal structure of a truncated form of the enzyme and demonstrate a severe decrease of cleavage efficiency when any of the nonbridging oxygen atoms upstream from the cleavage site was removed. Also observed was increased cleavage for oligonucleotides substituted with methylphosphonates downstream from the cleavage site. These effects were shown to be due primarily to alteration of the binding of the modified DNA substrates by human DNA topoisomerase I.     

Cobalt enhances DNA cleavage mediated by human topoisomerase II alpha in vitro and in cultured cells

Although cobalt is an essential trace element for humans, the metal is genotoxic and mutagenic at higher concentrations. Treatment of cells with cobalt generates DNA strand breaks and covalent protein-DNA complexes. However, the basis for these effects is not well understood. Since the toxic events induced by cobalt resemble those of topoisomerase II poisons, the effect of the metal on human topoisomerase IIalpha was examined. The level of enzyme-mediated DNA scission increased 6-13-fold when cobalt(II) replaced magnesium(II) in cleavage reactions. Cobalt(II) stimulated cleavage at all DNA sites observed in the presence of magnesium(II), and the enzyme cut DNA at several "cobalt-specific" sites. The increased level of DNA cleavage in the presence of cobalt(II) was partially due to a decrease in the rate of enzyme-mediated religation. Topoisomerase IIalpha retained many of its catalytic properties in reactions that included cobalt(II), including sensitivity to the anticancer drug etoposide and the ability to relax and decatenate DNA. Finally, cobalt(II) stimulated topoisomerase IIalpha-mediated DNA cleavage in the presence of magnesium(II) in purified systems and in human MCF-7 cells. These findings demonstrate that cobalt(II) is a topoisomerase II poison in vitro and in cultured cells and suggest that at least some of the genotoxic effects of the metal are mediated through topoisomerase IIalpha.     

Bacterial cell killing mediated by topoisomerase I DNA cleavage activity

DNA topoisomerases are important clinical targets for antibacterial and anticancer therapy. At least one type IA DNA topoisomerase can be found in every bacterium, making it a logical target for antibacterial agents that can convert the enzyme into poison by trapping its covalent complex with DNA. However, it has not been possible previously to observe the consequence of having such a stabilized covalent complex of bacterial topoisomerase I in vivo. We isolated a mutant of recombinant Yersinia pestis topoisomerase I that forms a stabilized covalent complex with DNA by screening for the ability to induce the SOS response in Escherichia coli. Overexpression of this mutant topoisomerase I resulted in bacterial cell death. From sequence analysis and site-directed mutagenesis, it was determined that a single amino acid substitution in the TOPRIM domain changing a strictly conserved glycine residue to serine in either the Y. pestis or E. coli topoisomerase I can result in a mutant enzyme that has the SOS-inducing and cell-killing properties. Analysis of the purified mutant enzymes showed that they have no relaxation activity but retain the ability to cleave DNA and form a covalent complex. These results demonstrate that perturbation of the active site region of bacterial topoisomerase I can result in stabilization of the covalent intermediate, with the in vivo consequence of bacterial cell death. Small molecules that induce similar perturbation in the enzyme-DNA complex should be candidates as leads for novel antibacterial agents.     

Minimal DNA requirement for topoisomerase II-mediated cleavage in vitro

The minimal DNA requirement for topoisomerase II-mediated DNA cleavage in vitro was determined by analyzing the interaction of the enzyme with sets of DNA substrates varying successively by single bases at the 5'- or 3'-end of either strand. A 16-base pair double-stranded region was established as the minimal duplex region required for topoisomerase II cleavage activity. The region was located symmetrically around the 4-base staggered cleavage site. Topoisomerase II-mediated cleavage within the 16-base pair core duplex, however, required single-stranded regions flanking the duplex to either the 5'- or 3'-sides, or an extension at both ends of the duplex with 1 or more base pairs.     

Calcium-promoted DNA cleavage by eukaryotic topoisomerase II: trapping the covalent enzyme-DNA complex in an active form

The effects of calcium ions on interactions between Drosophila melanogaster topoisomerase II and DNA were assessed. Although the divalent cation could not support DNA strand passage, it was able to promote high levels of enzyme-mediated DNA cleavage. Moreover, sites of cleavage on plasmid pBR322 generated in calcium-promoted reactions were similar to those obtained in the presence of magnesium. When calcium-containing enzyme-DNA mixtures were treated with ethylenediaminetetraacetic acid, cleaved nucleic acids could be generated in the absence of sodium dodecyl sulfate (SDS) or other denaturing detergents. The product of this SDS-independent calcium-promoted reaction was a covalent topoisomerase II-DNA complex. Enzyme molecules trapped in such complexes were found to be kinetically competent. Therefore, calcium should be a valuable tool for studying the enzymology of topoisomerase II mediated DNA cleavage.     

Specific DNA cleavage and binding by vaccinia virus DNA topoisomerase I

Cleavage of a defined linear duplex DNA by vaccinia virus DNA topoisomerase I was found to occur nonrandomly and infrequently. Approximately 12 sites of strand scission were detected within the 5372 nucleotides of pUC19 DNA. These sites could be classified as having higher or lower affinity for topoisomerase based on the following criteria. Higher affinity sites were cleaved at low enzyme concentration, were less sensitive to competition, and were most refractory to religation promoted by salt, divalent cations, and elevated temperature. Cleavage at lower affinity sites required higher enzyme concentration and was more sensitive to competition and induced religation. Cleavage site selection correlated with a pentameric sequence motif (C/T)CCTT immediately preceding the site of strand scission. Noncovalent DNA binding by topoisomerase predominated over covalent adduct formation, as revealed by nitrocellulose filter-binding studies. The noncovalent binding affinity of vaccinia topoisomerase for particular subsegments of pUC19 DNA correlated with the strength and/or the number of DNA cleavage sites contained therein. Thus, cleavage site selection is likely to be dictated by specific noncovalent DNA-protein interactions. This was supported by the demonstration that a mutant vaccinia topoisomerase (containing a Tyr----Phe substitution at the active site) that was catalytically inert and did not form the covalent intermediate, nevertheless bound DNA with similar affinity and site selectivity as the wild-type enzyme. Noncovalent binding is therefore independent of competence in transesterification. It is construed that the vaccinia topoisomerase is considerably more stringent in its cleavage and binding specificity for duplex DNA than are the cellular type I enzymes.     

Differential induction of Leishmania donovani bi-subunit topoisomerase I-DNA cleavage complex by selected flavones and camptothecin: activity of flavones against camptothecin-resistant topoisomerase I

Emergence of the bi-subunit topoisomerase I in the kinetoplastid family (Trypanosoma and Leishmania) has brought a new twist in topoisomerase research related to evolution, functional conservation and preferential sensitivities to the specific inhibitors of type IB topoisomerase family. In the present study, we describe that naturally occurring flavones baicalein, luteolin and quercetin are potent inhibitors of the recombinant Leishmania donovani topoisomerase I. These compounds bind to the free enzyme and also intercalate into the DNA at a very high concentration (300 µM) without binding to the minor grove. Here, we show that inhibition of topoisomerase I by these flavones is due to stabilization of topoisomerase I–DNA cleavage complexes, which subsequently inhibit the religation step. Their ability to stabilize the covalent topoisomerase I–DNA complex in vitro and in living cells is similar to that of the known topoisomerase I inhibitor camptothecin (CPT). However, in contrast to CPT, baicalein and luteolin failed to inhibit the religation step when the drugs were added to pre-formed enzyme substrate binary complex. This differential mechanism to induce the stabilization of cleavable complex with topoisomerase I and DNA by these selected flavones and CPT led us to investigate the effect of baicalein and luteolin on CPT-resistant mutant enzyme LdTOP1Δ39LS lacking 1–39 amino acids of the large subunit [B. B. Das, N. Sen, S. B. Dasgupta, A. Ganguly and H. K. Majumder (2005) J. Biol. Chem. 280, 16335–16344]. Baicalein and luteolin stabilize duplex oligonucleotide cleavage with LdTOP1Δ39LS. This observation was further supported by the stabilization of in vivo cleavable complex by baicalein and luteolin with highly CPT-resistant L.donovani strain. Taken together, our data suggest that the interacting amino acid residues of topoisomerase I may be partially overlapping or different for flavones and CPT. This study illuminates new properties of the flavones and provide additional insights into the ligand binding properties of L.donovani topoisomerase I.     

Exocyclic DNA lesions stimulate DNA cleavage mediated by human topoisomerase II alpha in vitro and in cultured cells

DNA adducts are mutagenic and clastogenic. Because of their harmful nature, lesions are recognized by many proteins involved in DNA repair. However, mounting evidence suggests that lesions also are recognized by proteins with no obvious role in repair processes. One such protein is topoisomerase II, an essential enzyme that removes knots and tangles from the DNA. Because topoisomerase II generates a protein-linked double-stranded DNA break during its catalytic cycle, it has the potential to fragment the genome. Previous studies indicate that abasic sites and other lesions that distort the double helix stimulate topoisomerase II-mediated DNA cleavage. Therefore, to further explore interactions between DNA lesions and the enzyme, the effects of exocyclic adducts on DNA cleavage mediated by human topoisomerase IIalpha were determined. When located within the four-base overhang of a topoisomerase II cleavage site (at the +2 or +3 position 3' relative to the scissile bond), 3,N(4)-ethenodeoxycytidine, 3,N(4)-etheno-2'-ribocytidine, 1,N(2)-ethenodeoxyguanosine, pyrimido[1,2-a]purin-10(3H)-one deoxyribose (M(1)dG), and 1,N(2)-propanodeoxyguanosine increased DNA scission approximately 5-17-fold. Enhanced cleavage did not result from an increased affinity of topoisomerase IIalpha for adducted DNA or a decreased rate of religation. Therefore, it is concluded that these exocyclic lesions act by accelerating the forward rate of enzyme-mediated DNA scission. Finally, treatment of cultured human cells with 2-chloroacetaldehyde, a reactive metabolite of vinyl chloride that generates etheno adducts, increased cellular levels of DNA cleavage by topoisomerase IIalpha. This finding suggests that type II topoisomerases interact with exocyclic DNA lesions in physiological systems.     

DNA topoisomerase I from calf thymus is inhibited in vitro by poly(ADP-ribosylation)

A slight DNA topoisomerase I activity was detected in highly purified poly(ADP-Rib)polymerase prepared from calf thymus. This copurified activity was found to be suppressed under conditions where the poly(ADP-ribosylation) reaction occurs in the presence of NAD. Purified topoisomerase I from calf thymus was shown to be ADP-ribosylated by poly(ADP-Rib) polymerase purified from the same tissue. Poly(ADP-ribosylation) of topoisomerase I produces an inhibition of the enzymatic activity in parallel to the extent of ADP-ribosylation. The fact that a slight poly(ADP-Rib) polymerase activity was also found to copurify with a topoisomerase I preparation and that topoisomerase I activity can be modified by ADP-ribosylation, may suggest a spatial and functional correlation of these two enzymes in chromatin.     

Minimal DNA duplex requirements for topoisomerase I-mediated cleavage in vitro

The minimal DNA duplex requirements for topoisomerase I-mediated cleavage at a specific binding sequence were determined by analyzing the interaction of the enzyme with sets of DNA substrates varying successively by single nucleotides at the 5'- or 3' end of either strand. Topoisomerase I cleavage experiments showed a minimal region of nine nucleotides on the scissile strand and five nucleotides on the noncleaved strand. On the scissile strand, seven of the nine nucleotides were situated upstream to the cleavage site, while all five nucleotides required on the non-cleaved strand were located to this side. The results suggested that topoisomerase I bound tightly to this region, stabilizing the DNA duplex extensively. On minimal substrates which were partially single-stranded downstream to the cleavage site, cleavage was suicidal, that is, the enzyme was able to cleave the substrates, but unable to perform the final religation.     

In vivo sequencing of camptothecin-induced topoisomerase I cleavage sites in human colon carcinoma cells.

Camptothecin (CPT) is a specific topoisomerase I (top1) poison which traps top1 cleavable complexes; e.g. top1-linked DNA single-strand breaks with 5'-hydroxyl and 3'-top1 linked termini. CPT is also a potent anticancer agent and several of its derivatives have recently shown activity in the chemotherapy of solid tumors. Our aim was to apply the ligation-mediated polymerase chain reaction (LM-PCR) method to DNA extracted from CPT-treated cells in order to: (i) evaluate LM-PCR as a sensitive technique to detect in vivo CPT-induced cleavable complexes; (ii) investigate the frequency and distribution of CPT-induced DNA damage in vivo ; and (iii) compare the distribution and intensity of cleavage sites in vivo and in vitro. This report describes a protocol allowing the sequencing of top1-mediated DNA strand breaks induced by CPT in the coding strand of the 18S rRNA gene of human colon carcinoma cells. CPT or its clinical derivatives, topotecan, CPT-11, SN-38, and 9-aminocamptothecin differed in their potency and exhibited differences in their DNA cleavage pattern, which is consistent with our previous in vitro studies [Tanizawa et al . (1995) Biochemistry , 43, 7200-7206]. CPT-induced DNA cleavages induced in the presence of purified top1 were induced at the same sites in the human 18S rDNA. However, the relative intensity of the cleavages were different in vivo and in vitro. Because mammalian cells contain approximately 300 copies of the rDNA gene per genome, rDNA could be used to monitor CPT-induced DNA cleavage in different cell lines and possibly in tumor samples.     

The geometry of DNA supercoils modulates the DNA cleavage activity of human topoisomerase I

Human topoisomerase I plays an important role in removing positive DNA supercoils that accumulate ahead of replication forks. It also is the target for camptothecin-based anticancer drugs that act by increasing levels of topoisomerase I-mediated DNA scission. Evidence suggests that cleavage events most likely to generate permanent genomic damage are those that occur ahead of DNA tracking systems. Therefore, it is important to characterize the ability of topoisomerase I to cleave positively supercoiled DNA. Results confirm that the human enzyme maintains higher levels of cleavage with positively as opposed to negatively supercoiled substrates in the absence or presence of anticancer drugs. Enhanced drug efficacy on positively supercoiled DNA is due primarily to an increase in baseline levels of cleavage. Sites of topoisomerase I-mediated DNA cleavage do not appear to be affected by supercoil geometry. However, rates of ligation are slower with positively supercoiled substrates. Finally, intercalators enhance topoisomerase I-mediated cleavage of negatively supercoiled substrates but not positively supercoiled or linear DNA. We suggest that these compounds act by altering the perceived topological state of the double helix, making underwound DNA appear to be overwound to the enzyme, and propose that these compounds be referred to as ‘topological poisons of topoisomerase I’.     

Recombinogenic flap ligation mediated by human topoisomerase I

Aberration of eukaryotic topoisomerase I catalysis leads to potentially recombinogenic pathways by allowing the joining of heterologous DNA strands. Recently, a new ligation pathway (flap ligation) was presented for vaccinia virus topoisomerase I, in which blunt end cleavage complexes ligate the recessed end of duplex acceptors having a single-stranded 3'-tail. This reaction was suggested to play an important role in the repair of topoisomerase I-induced DNA double-strand breaks. Here, we characterize flap ligation mediated by human topoisomerase I. We demonstrate that cleavage complexes containing the enzyme at a blunt end allow invasion of a 3'-acceptor tail matching the scissile strand of the donor, which facilitates ligation of the recessed 5'-hydroxyl end. However, the reaction was strictly dependent on the length of double-stranded DNA of the donor complexes, and longer stretches of base-pairing inhibited strand invasion. The stabilization of the DNA helix was most probably provided by the covalently bound enzyme itself, since deleting the N-terminal domain of human topoisomerase I stimulated flap ligation. We suggest that stabilization of the DNA duplex upon enzyme binding may play an important role during normal topoisomerase I catalysis by preventing undesired strand transfer reactions. For flap ligation to function in a repair pathway, factors other than topoisomerase I, such as helicases, would be necessary to unwind the DNA duplex and allow strand invasion.     

SOS induction by stabilized topoisomerase IA cleavage complex occurs via the RecBCD pathway

Accumulation of mutant topoisomerase I cleavage complex can lead to SOS induction and cell death in Escherichia coli. The single-stranded break associated with mutant topoisomerase I cleavage complex is converted to double-stranded break, which then is processed by the RecBCD pathway, followed by association of RecA with the single-stranded DNA.