Binding of ATP to human DNA topoisomerase I resulting in an alteration of the conformation of the enzyme.

It has been known for some time that ATP inhibits the DNA relaxation activity of human DNA topoisomerase I. However, the underlying mechanism of this inhibitory effect remains largely unknown. Using filter binding assays, the binding of human DNA topoisomerase I to DNA was decreased in the presence of ATP. This result suggests that the inhibition of DNA relaxation activity of human DNA topoisomerase I by ATP is at the binding step rather than at the nicking or resealing step. DNA topoisomerase I cleavage assay further supports this notion. ATP-agarose binding and UV cross-linking assays also demonstrate that ATP directly and specifically binds human DNA topoisomerase I. To address whether the ATP binding results in conformational changes in human DNA topoisomerase I, various proteases were employed for detecting potential protein conformational changes. Our results indicated that the proteolytic susceptibilities of trypsin and chymotrypsin were altered in the presence of ATP. The result suggests that the conformation of human DNA topoisomerase I was altered upon ATP binding. In addition, the binding between ATP and human DNA topoisomerase I was also reduced by increasing concentrations of DNA. Our data suggests that human DNA topoisomerase I exhibits at least two incompatible conformations. One conformation is in the form of a topoisomerase I-ATP complex, which inhibits DNA relaxation activity of human DNA topoisomerase I, and the other, a topoisomerase I-DNA complex, which exerts DNA relaxation activity. Our studies identify the role of ATP in the regulation of human DNA topoisomerase I and provide a substantial implication of how human DNA topoisomerase I compromises its versatile functions.     

Topoisomerase activity is associated with purified SV40 T antigen.

Purified SV40 T antigen has been assayed for topoisomerase activity. The ability to relax negatively-supercoiled SV40 DNA was found in preparations of T antigen purified either from human 293 cells infected with Ad5-SVR111 virus or from insect Sf9 cells infected with recombinant baculovirus 941T. The T antigen-associated relaxing activity was stimulated by MgCl2 and was not dependent on ATP, suggesting that it is not due to cellular topoisomerase II. The topoisomerase activity was immunoprecipitated by a monoclonal antibody specific for T antigen, but not by a control monoclonal antibody. In addition, immunoblotting of purified T antigen from human 293 cells with antihuman topoisomerase I and anti-human topoisomerase II antibodies failed to detect cellular topoisomerases I or II. Sedimentation analysis of purified T antigen revealed that the topoisomerase activity co-sedimented with the hexameric form of T antigen at 23S. The topoisomerase activity is, therefore, either inherent to T antigen or due to a cellular topoisomerase I tightly bound to, and co-purifying with, T antigen.     

Cloning and characterization of the gene for the somatic form of DNA topoisomerase I from Xenopus laevis.

Two distinct tissue-specific forms of DNA topoisomerase I with M(r) of 165 and 110 kDa have been purified from oocytes and somatic cells respectively of the African frog Xenopus laevis. In this paper, cDNAs encoding a Xenopus topoisomerase I were cloned using PCR primers derived from sequences of yeast and human topoisomerase I. A polypeptide expressed from a portion of the coding sequence was recognized by an antiserum directed against the somatic topoisomerase I that had previously been shown to be unable to cross-react with the oocyte enzyme. Thus, the clone encodes the somatic cell topoisomerase I. An antiserum raised against a synthetic peptide containing the sequence surrounding the active site tyrosine of the somatic topoisomerase I reacts with the enzymes purified from both oocytes and somatic cells, indicating that the two enzymes share some limited sequence homology. RNA blot hybridization showed that oocytes contain an abundant store of somatic topoisomerase I mRNA that is not efficiently polyadenylated in oocytes. This stored RNA contains a consensus cytoplasmic polyadenylation element that is found in a variety of mRNAs that are translationally repressed in oocytes. Microinjection into oocytes of in vitro transcribed mRNA prepared from a Myc-tagged construct of the somatic topoisomerase I sequence is translated to yield a 110 kDa product. This suggests that the oocyte-specific 165 kDa topoisomerase I is not produced by tissue-specific post-translational modification of the somatic topoisomerase I. The oocyte enzyme appears to be produced from a minor mRNA species in oocytes that has not yet been identified.     

The RNA splicing factor ASF/SF2 inhibits human topoisomerase I mediated DNA relaxation

Human topoisomerase I interacts with and phosphorylates the SR-family of RNA splicing factors, including ASF/SF2, and has been suggested to play an important role in the regulation of RNA splicing. Here we present evidence to support the theory that the regulation can go the other way around with the SR-proteins controlling topoisomerase I DNA activity. We demonstrate that the splicing factor ASF/SF2 inhibits relaxation by interfering with the DNA cleavage and/or DNA binding steps of human topoisomerase I catalysis. The inhibition of relaxation correlated with the ability of various deletion mutants of the two proteins to interact directly, suggesting that an interaction between the RS-domain of ASF/SF2 and a region between amino acid residues 208-735 on topoisomerase I accounts for the observed effect. Consistently, phosphorylation of the RS-domain with either topoisomerase I or a human cell extract reduced the inhibition of relaxation activity. Taken together with the previously published studies of the topoisomerase I kinase activity, these observations suggest that topoisomerase I activity is shifted from relaxation to kinasing by specific interaction with SR-splicing factors.     

Stimulated activity of human topoisomerases IIalpha and IIbeta on RNA-containing substrates.

Eukaryotic topoisomerase II is a dimeric nuclear enzyme essential for DNA metabolism and chromosome dynamics. Central to the activities of the enzyme is its ability to introduce transient double-stranded breaks in the DNA helix, where the two subunits of the enzyme become covalently attached to the generated 5'-ends through phosphotyrosine linkages. Here, we demonstrate that human topoisomerases IIalpha and IIbeta are able to cleave ribonucleotide-containing substrates. With suicide substrates, which are partially double-stranded molecules containing a 5'-recessed strand, cleavage of both strands was stimulated approximately 8-fold when a ribonucleotide rather than a deoxyribonucleotide was present at the scissile phosphodiester of the recessed strand. The existence of a ribonucleotide at the same position in a normal duplex substrate also enhanced topoisomerase II-mediated cleavage, although to a lesser extent. The enzyme covalently linked to the 5'-ribonucleotide in the cleavage complex efficiently performed ligation, and ligation occurred equally well to acceptor molecules terminated by either a 3'-ribo- or deoxyribonucleotide. Besides the enhanced topoisomerase II-mediated cleavage of ribonucleotide-containing substrates, cleavage of such substrates could be further stimulated by ATP or antitumor drugs. In conclusion, the observed in vitro activities of the human topoisomerase II isoforms indicate that the enzymes can operate on RNA or RNA-containing substrates and thus might possess an intrinsic RNA topoisomerase activity, as has previously been demonstrated for Escherichia coli topoisomerase III.     

On the role of E-ring oxygen atoms in the binding of camptothecin to the topoisomerase I-DNA covalent binary complex

A recent X-ray crystallographic analysis of the binding of a water soluble camptothecin analogue to the human topoisomerase I-DNA covalent binary complex has suggested the existence of some novel features in the way that camptothecin is bound to the binary complex. Four additional models based on chemical and biochemical data have also been proposed. Presently we describe S-containing analogues of camptothecin prepared on the basis of these models, and report their ability to form stable ternary complexes with human topoisomerase I, and to mediate cytotoxicity at the locus of topoisomerase I. The results indicate that replacement of the 20-OH group of CPT with a SH functionality results in diminution of the potency of CPT as a topoisomerase I poison, while replacement of the O atoms at positions 20 and 21 with S atoms results in essentially complete loss of topoisomerase I inhibitory activity.     

Escherichia coli DNA topoisomerase III: purification and characterization of a new type I enzyme

A new topoisomerase capable of relaxing negatively supercoiled DNA in Escherichia coli has been identified during chromatography on novobiocin-Sepharose. A simple and reproducible purification procedure is described to obtain this enzyme, called topoisomerase III (topo III), in a homogeneous form. The protein is a single polypeptide with a molecular weight of 74 000 +/- 2000 and is a type I topoisomerase, changing the linking number of DNA circles in steps of one. It is present in deletion strains lacking the topA gene and further differs from the well-studied topoisomerase I (omega protein; Eco topo I) in (1) its requirement for K+ in addition to Mg2+ to exhibit optimal activity and (2) its affinity to novobiocin-Sepharose. Positively supercoiled DNA is not relaxed during exposure to the enzyme. Topo III has no ATPase activity, and ATP does not show any discernible effect on the reduction of superhelical turns. The purified topoisomerase has no supercoiling activity and is unaffected by high concentrations of oxolinic acid and novobiocin in the relaxing reaction. Single-stranded DNA and spermidine strongly inhibit the topoisomerase activity.     

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.     

DNA sequence selectivity of human topoisomerase I‐mediated DNA cleavage induced by camptothecin

In probing the mechanism of inhibition of hypoxia inducible factor (HIF-1) by campothecins, we investigated the ability of human topoisomerase I to bind and cleave HIF-1 response element (HRE), which contains the known camptothecin-mediated topoisomerase I cleavage site 5′-TG. We observed that the selection of 5′-TG by human topoisomerase I and topotecan depends to a large extent on the specific flanking sequences, and that the presence of a G at the −2 position (where cleavage occurs between −1 and +1) prevents the HRE site from being a preferred site for such cleavage. Furthermore, the presence of −2 T/A can induce the cleavage at a less preferred TC or TA site. However, in the absence of a more preferred site, the HRE site is shown to be cleaved by human topoisomerase I in the presence of topotecan. Thus, it is implied that the −2 base has a significant influence on the selection of the camptothecin-mediated Topo I cleavage site, which can overcome the preference for +1G. While the cleavage site recognition has been known to be based on the concerted effect of several bases spanning the cleavage site, such a determining effect of an individual base has not been previously recognized. A possible base-specific interaction between DNA and topoisomerase I may be responsible for this sequence selectivity.     

The C-terminal domain but not the tyrosine 723 of human DNA topoisomerase I active site contributes to kinase activity.

Human DNA topoisomerase I not only has DNA relaxing activity, but also splicing factors phosphorylating activity. Topo I shows strong preference for ATP as the phosphate donor. We used photoaffinity labeling with the ATP analogue [alpha-32P] 8-azidoadenosine-5'-triphosphate combined with limited proteolysis to characterize Topo I domains involved in ATP binding. The majority of incorporated analogue was associated with two fragments derived from N-terminal and C-terminal regions of Topo I, respectively. However, mutational analysis showed that deletion of the first 138 N-terminal residues, known to be dispensable for topoisomerase activity, did not change the binding of ATP or the kinase activity. In contrast, deletion of 162 residues from the C-terminal domain was deleterious for ATP binding, kinase and topoisomerase activities. Furthermore, a C-terminal tyrosine 723 mutant lacking topoisomerase activity is still able to bind ATP and to phosphorylate SF2/ASF, suggesting that the two functions of Topo I can be separated. These findings argue in favor of the fact that Topo I is a complex enzyme with a number of potential intra-cellular functions.     

Active DNA topoisomerase IIalpha is a component of the salt-stable centrosome core

Recently, we reported that the monoclonal antibody specific for human DNA topoisomerase IIalpha, Ki-S1, stains not only the nuclei of human A431 cells but also extranuclear structures suggestive of centrosomes (Meyer, K. N., Kjeldsen, E., Straub, T., Knudsen, B. K., Kikuchi, A., Hickson, I. D., Kreipe, H., and Boege, F. (1997) J. Cell Biol. 136, 775-788). Here, we confirm colocalization of Ki-S1 with the centrosomal marker gamma-tubulin. In addition, we show labeling of centrosomes by peptide antibodies against the N and C termini of human topoisomerase IIalpha. Probing Western blots of isolated centrosomes with topoisomerase IIalpha antibodies, we demonstrate a protein band of 170 kDa. Moreover, isolated centrosomes exhibited DNA decatenation and relaxation activity correlated to the amount of topoisomerase IIalpha protein in the same way as seen in the pure recombinant enzyme. Topoisomerase IIalpha epitopes could not be removed from centrosomes by salt extraction, DNase treatment, or RNase treatment, procedures that completely removed the enzyme from nuclei. Taken together, these observations suggest that active topoisomerase IIalpha is bound tightly to the centrosome in a DNA-independent manner. Because such centrosomal topoisomerase IIalpha was also present in quiescent lymphocytes devoid of topoisomerase IIalpha in the nuclei, we assume that it might be a long-lived storage form.     

DNA supercoiling during ATP-dependent DNA translocation by the type I restriction enzyme EcoAI

Type I restriction enzymes cleave DNA at non-specific sites far from their recognition sequence as a consequence of ATP-dependent DNA translocation past the enzyme. During this reaction, the enzyme remains bound to the recognition sequence and translocates DNA towards itself simultaneously from both directions, generating DNA loops, which appear to be supercoiled when visualised by electron microscopy. To further investigate the mechanism of DNA translocation by type I restriction enzymes, we have probed the reaction intermediates with DNA topoisomerases. A DNA cleavage-deficient mutant of EcoAI, which has normal DNA translocation and ATPase activities, was used in these DNA supercoiling assays. In the presence of eubacterial DNA topoisomerase I, which specifically removes negative supercoils, the EcoAI mutant introduced positive supercoils into relaxed plasmid DNA substrate in a reaction dependent on ATP hydrolysis. The same DNA supercoiling activity followed by DNA cleavage was observed with the wild-type EcoAI endonuclease. Positive supercoils were not seen when eubacterial DNA topoisomerase I was replaced by eukaryotic DNA topoisomerase I, which removes both positive and negative supercoils. Furthermore, addition of eukaryotic DNA topoisomerase I to the product of the supercoiling reaction resulted in its rapid relaxation. These results are consistent with a model in which EcoAI translocation along the helical path of closed circular DNA duplex simultaneously generates positive supercoils ahead and negative supercoils behind the moving complex in the contracting and expanding DNA loops, respectively. In addition, we show that the highly positively supercoiled DNA generated by the EcoAI mutant is cleaved by EcoAI wild-type endonuclease much more slowly than relaxed DNA. This suggests that the topological changes in the DNA substrate associated with DNA translocation by type I restriction enzymes do not appear to be the trigger for DNA cleavage.