Eukaryotic type I topoisomerase is enriched in the nucleolus and catalytically active on ribosomal DNA.

The distribution of eukaryotic DNA topoisomerase I in the cell has been analyzed at four levels: (i) at the level of the nuclear matrix; (ii) at the cytological level by immunofluorescence of whole cells; (iii) at the electron microscopic level using the protein A/colloidal gold technique; and (iv) at the level of DNA to identify in situ the sequence upon which topoisomerase I is catalytically active. Although topoisomerase I is clearly distributed non-randomly in the nucleus, the unique distribution of the enzyme is not related to the nuclear matrix. The data support the conclusion that topoisomerase I is heavily concentrated in the nucleolus of the cell; furthermore, particular regions within the nucleolus are depleted of topoisomerase. A technique has been developed which allows isolation and analysis of the cellular DNA sequences covalently attached to topoisomerase. Ribosomal DNA sequences are at least 20-fold enriched in topoisomerase/DNA complexes isolated directly from a chromosomal setting, relative to total DNA. This is the first direct evidence that topoisomerase I is catalytically active on ribosomal DNA in vivo.     

Eukaryotic topoisomerase II cleavage of parallel stranded DNA tetraplexes.

A guanine-rich single-stranded DNA from the human immunoglobulin switch region was shown by Sen and Gilbert [Nature, (1988) 334, 364-366] to be able to self-associate to form a stable four-stranded parallel DNA structure. Topoisomerase II did not cleave the single-stranded DNA molecule. Surprisingly, the enzyme did cleave the same DNA sequence when it was annealed into the four-stranded structure. The two cleavage sites observed were the same as those found when this DNA molecule was paired with a complementary molecule to create a normal B-DNA duplex. These cleavages were shown to be protein-linked and reversible by the addition of salt, suggesting a normal topoisomerase II reaction mechanism. In addition, an eight-stranded DNA molecule created by the association of a complementary oligonucleotide with the four-stranded structure was also cleaved by topoisomerase II despite being resistant to restriction endonuclease digestion. These results suggest that a single strand of DNA may possess the sequence information to direct topoisomerase II to a binding site, but the site must be base paired in a proper manner to do so. This demonstration of the ability of a four-stranded DNA molecule to be a substrate for an enzyme further suggests that these DNA structures may be present in cells.     

Eukaryotic topoisomerase I-DNA interaction is stabilized by helix curvature.

The influence of DNA structure on topoisomerase I-DNA interaction has been investigated using a high affinity binding site and mutant derivatives thereof. Parallel determinations of complex formation and helix structure in the absence of superhelical stress suggest that the interaction is intensified by stable helix curvature. Previous work showed that a topoisomerase I binding site consists of two functionally distinct subdomains. A region located 5' to the topoisomerase I cleavage site is essential for binding. The region 3' to the cleavage site is covered by the enzyme, but not essential. We report here that the helix conformation of the latter region is an important modulator of complex formation. Thus, complex formation is markedly stimulated, when an intrinsically bent DNA segment is installed in this region. A unique pattern of phosphate ethylation interferences in the 3'-part of the binding site indicates that sensing of curvature involves backbone contacts. Since dynamic curvature in supercoiled DNA may substitute for stable curvature, our findings suggest that topoisomerase I is able to probe DNA topology by assessment of writhe, rather than twist.     

Radical-induced purine lesion formation is dependent on DNA helical topology

Abstract

Herein we report the quantification of purine lesions arising from gamma-radiation sourced hydroxyl radicals (HO^?) on tertiary dsDNA helical forms of supercoiled (SC), open circular (OC), and linear (L) conformation, along with single-stranded folded and non-folded sequences of guanine-rich DNA in selected G-quadruplex structures. We identify that DNA helical topology and folding plays major, and unexpected, roles in the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxo-dA), along with tandem-type purine lesions 5′,8-cyclo-2′-deoxyguanosine (5′,8-cdG) and 5′,8-cyclo-2′-deoxyadenosine (5′,8-cdA). SC, OC, and L dsDNA conformers together with folded and non-folded G-quadruplexes d[TGGGGT]₄ (TG4T), d[AGGG(TTAGGG)₃] (Tel22), and the mutated tel24 d[TTGGG(TTAGGG)₃A] (mutTel24) were exposed to HO^? radicals and purine lesions were then quantified via stable isotope dilution LC-MS/MS analysis. Purine oxidation in dsDNA follows L?>?OC???SC indicating greater damage towards the extended B-DNA topology. Conversely, G-quadruplex sequences were significantly more resistant toward purine oxidation in their unfolded states as compared with G-tetrad folded topologies; this effect is confirmed upon comparative analysis of Tel22 (～50% solution folded) and mutTel24 (～90% solution folded). In an effort to identify the accessibly of hydroxyl radicals to quadruplex purine nucleobases, G-quadruplex solvent cavities were then modeled at 1.33?Å with evidence suggesting that folded G-tetrads may act as potential oxidant traps to protect against chromosomal DNA damage.     

DNA topology: feeling the pulse of a topoisomerase

The action of individual type II DNA topoisomerases has been followed in real time by observing the elastic response of single DNA molecules to sequential strand passage events. Micromanipulation methods provide a complementary approach to biochemical studies for investigating the mechanism of DNA topoisomerases.     

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.     

Eukaryotic topoisomerases recognize nucleic acid topology by preferentially interacting with DNA crossovers.

Eukaryotic topoisomerases recognize DNA topology and preferentially react with positively or negatively supercoiled molecules over relaxed substrates. To elucidate the mechanism of this recognition, we examined the interaction of topoisomerases with DNA by electron microscopy. Under all conditions employed, approximately 90% of the bound type I or II enzyme was observed at points of helix--helix juxtaposition on negatively supercoiled plasmids which contained as few as four crossovers. Recognition was independent of torsional stress, as enzyme molecules were also found at crossovers on linear DNA. Since juxtaposed helices are more prevalent in supercoiled compared with relaxed nucleic acids, we propose that eukaryotic topoisomerases I and II recognize underwound or overwound substrates by interacting preferentially with DNA crossovers. This may represent a general mechanism for the recognition of DNA topology by proteins.     

Simian virus 40 DNA replication is dependent on an interaction between topoisomerase I and the C-terminal end of T antigen

Topoisomerase I (topo I) is needed for efficient initiation of simian virus 40 (SV40) DNA replication and for the formation of completed DNA molecules. Two distinct binding sites for topo I have been previously mapped to the N-terminal (residues 83 to 160) and C-terminal (residues 602 to 708) regions of T antigen. By mutational analysis, we identified a cluster of six residues on the surface of the helicase domain at the C-terminal binding site that are necessary for efficient binding to topo I in enzyme-linked immunosorbent assay and far-Western blot assays. Mutant T antigens with single substitutions of these residues were unable to participate normally in SV40 DNA replication. Some mutants were completely defective in supporting DNA replication, and replication was not enhanced in the presence of added topo I. The same mutants were the ones that were severely compromised in binding topo I. Other mutants demonstrated intermediate levels of activity in the DNA replication assay and were correspondingly only partially defective in binding topo I. Mutations of nearby residues outside this cluster had no effect on DNA replication or on the ability to bind topo I. These results strongly indicate that the association of topo I with these six residues in T antigen is essential for DNA replication. These residues are located on the back edges of the T-antigen double hexamer. We propose that topo I binds to one site on each hexamer to permit the initiation of SV40 DNA replication.     

Conformational changes in E. coli DNA topoisomerase I.

DNA topoisomerases are the enzymes responsible for maintaining the topological states of DNA. In order to change the topology of DNA, topoisomerases pass one or two DNA strands through transient single or double strand breaks in the DNA phosphodiester backbone. It has been proposed that both type IA and type II enzymes change conformation dramatically during the reaction cycle in order to accomplish these transformations. In the case of Escherichia coli DNA topoisomerase I, it has been suggested that a 30 kDa fragment moves away from the rest of the protein to create an entrance into the central hole in the protein. Structures of the 30 kDa fragment reveal that indeed this fragment can change conformation significantly. The fragment is composed of two domains, and while the domains themselves remain largely unchanged, their relative arrangement can change dramatically.     

Histone H1 inhibits eukaryotic DNA topoisomerase I.

Histone H1 inhibits the catalytic activity of topoisomerase I in vitro. The relaxation activity of the enzyme is partially inhibited at a molar ratio of one histone H1 molecule per 40 base pairs (bp) of DNA and completely inhibited at a molar ratio of one histone H1 molecule per 10 base pairs of DNA. Increasing the amount of enzyme at a constant histone H1 to DNA ratio antagonizes the inhibition. This indicates that topoisomerase I and histone H1 compete for binding sites on the substrate DNA molecules. Consistent with this we show on the sequence level that histone H1 inhibits the cleavage reaction of topoisomerase I on linear DNA fragments.     

Protein-nucleotide interactions in E. coli DNA topoisomerase I.

DNA topoisomerases are the enzymes responsible for controlling and maintaining the topological states of DNA. Type IA enzymes work by transiently breaking the phosphodiester backbone of one strand to allow passage of another strand through the break. The protein has to perform complex rearrangements of the DNA, and hence it is likely that different regions of the enzyme bind DNA with different affinities. In order to identify some of the DNA binding sites in the protein, we have solved the structures of several complexes of the 67 kDa N-terminal fragment of Escherichia coli DNA topoisomerase I with mono- and trinucleotides. There are five different binding sites in the complexes, one of which is adjacent to the active site. Two other sites are in the central hole of the protein and may represent general DNA binding regions. The positions of these sites allow us to identify different DNA binding regions and to understand their possible roles in the catalytic cycle.     

Eukaryotic topoisomerase II. Characterization of enzyme turnover

While the binding of adenyl-5'-yl imidodiphosphate (App(NH)p) to Drosophila melanogaster topoisomerase II induces a double-stranded DNA passage reaction, its nonhydrolyzable beta,gamma-imidodiphosphate bond prevents enzyme turnover (Osheroff, N., Shelton, E. R., and Brutlag, D. L. (1983) J. Biol. Chem. 258, 9536-9543). Therefore, this ATP analog was used to characterize the interactions between Drosophila topoisomerase II and DNA which occur after DNA strand passage but before enzyme turnover. In the presence of App(NH)p, a stable post-strand passage topoisomerase II-nucleic acid complex is formed when circular DNA substrates are employed. Although noncovalent in nature, these complexes are resistant to increases in ionic strength and show less than 5% dissociation under salt concentrations (greater than 500 mM) that disrupt 95% of the enzyme-DNA interactions formed in the absence of App(NH)p or under a variety of other conditions that do not support DNA strand passage. These results strongly suggest that the process of enzyme turnover not only regenerates the active conformation of topoisomerase II but also confers upon the enzyme the ability to disengage from its nucleic acid product. Experiments with linear DNA molecules indicate that after strand passage has taken place, topoisomerase II may be able to travel along its DNA substrate by a linear diffusion process that is independent of enzyme turnover. Further studies demonstrate that the regeneration of the enzyme's catalytic center does not require enzyme turnover, since topoisomerase II can cleave double-stranded DNA substrates after strand passage has taken place. Finally, while the 2'-OH and 3'-OH of ATP are important for its interaction with Drosophila topoisomerase II, neither are required for turnover.     

The 165-kDa DNA topoisomerase I from Xenopus laevis oocytes is a tissue-specific variant.

Two forms of topoisomerase I can be purified from Xenopus laevis. A protein with a molecular mass of 165 kDa has been identified as topoisomerase I in ovaries (Richard and Bogenhagen, 1989. J. Biol. Chem. 264, 4704-4709). When a similar purification is performed using liver tissue, topoisomerase I is purified as a 110-kDa protein. Separate rabbit antisera were raised against oocyte and liver topoisomerase I polypeptides. Each antiserum reacts in immunoblotting or immunoprecipitation procedures only with the tissue-specific topoisomerase I polypeptide against which it was generated. The failure of the antiserum raised against liver topoisomerase I to cross-react with the oocyte enzyme suggests that the smaller topoisomerase I is not derived from the 165-kDa oocyte enzyme by proteolysis. X. laevis tissue culture cells lysed and processed in the presence of SDS contain the 110-kDa form of topoisomerase I. The 165-kDa form of topoisomerase I disappears during oocyte maturation in vitro.     

Iridoids as DNA topoisomerase I poisons

The discovery of new topoisomerase I inhibitors is necessary since most of the antitumor drugs are targeted against type II and only a very few can specifically affect type I. Topoisomerase poisons generate toxic DNA damage by stabilization of the covalent DNA-topoisomerase cleavage complex and some have therapeutic efficacy in human cancer. Two iridoids, aucubin and geniposide, have shown antitumoral activities, but their activity against topoisomerase enzymes has not been tested. Here it was found that both compounds are able to stabilize covalent attachments of the topoisomerase I subunits to DNA at sites of DNA strand breaks, generating cleavage complexes intermediates so being active as poisons of topoisomerase I, but not topoisomerase II. This result points to DNA damage induced by topoisomerase I poisoning as one of the possible mechanisms by which these two iridoids have shown antitumoral activity, increasing interest in their possible use in cancer chemoprevention and therapy.     

Chloroplast DNA topoisomerase I from cauliflower

An ATP-independent DNA topoisomerase has been isolated from chloroplasts of cauliflower leaves (Brassica oleracea var. botrytis) through DEAE-cellulose, AF-blue Toyopearl, and hydroxyapatite column chromatography. The sedimentation coefficient and Stokes radius of this enzyme are 3.6S and 3.6 nm, respectively, and the molecular weight of native enzyme is estimated to be 54,000. This enzyme changes the linking number in steps of one. The enzyme activity is stimulated by MgCl2, and this enzyme shows optimum activity at 30 degrees C in the range of 3 mM MgCl2 + 100 mM KCl-10 mM MgCl2 + 50 mM KCl. The enzyme activity was reduced remarkably by N-ethylmaleimide, indicating that a free sulfhydryl group is important for the activity; heparin and ellipticine also reduced the activity. Both cauliflower chloroplast topoisomerase and spinach chloroplast topoisomerase can relax positive supercoils as well as negative supercoils. From these properties, cauliflower chloroplast topoisomerase can be classified as a eukaryotic type I DNA topoisomerase.     

Mouse genes encoding DNA topoisomerase I

We have initiated a genetic analysis of the physiologically important enzyme type I DNA topoisomerase in mouse. The exon-intron structures of the 5 part and the 3 part of the active gene, Top-1, were determined and shown to be quite similar to those of the previously determined human gene TOP1. The active mouse gene was mapped to the distal Chromosome (Chr) 2. In addition, the mouse genome contains one truncated processed topoisomerase-I-related pseudogene (retroposon), Top-1ps, on Chr 16. The Top-1ps locus, together with the immunoglobulin-lambda-light-chain locus, defines and additional conserved linkage group common to murine Chr 16 and human Chr 22, the site of the human pseudogene TOP1P2. The mapping data suggest that the pseudogene was established before mammalian radiation. Structural features, shared by the mouse and the human pseudogene, support this possibility.