Residues within the N-terminal domain of human topoisomerase I play a direct role in relaxation.

All eukaryotic forms of DNA topoisomerase I contain an extensive and highly charged N-terminal domain. This domain contains several nuclear localization sequences and is essential for in vivo function of the enzyme. However, so far no direct function of the N-terminal domain in the in vitro topoisomerase I reaction has been reported. In this study we have compared the in vitro activities of a truncated form of human topoisomerase I lacking amino acids 1-206 (p67) with the full-length enzyme (p91). Using these enzyme forms, we have identified for the first time a direct role of residues within the N-terminal domain in modulating topoisomerase I catalysis, as revealed by significant differences between p67 and p91 in DNA binding, cleavage, strand rotation, and ligation. A comparison with previously published studies showing no effect of deleting the first 174 or 190 amino acids of topoisomerase I (Stewart, L., Ireton, G. C., and Champoux, J. J. (1999) J. Biol. Chem. 274, 32950-32960; Bronstein, I. B., Wynne-Jones, A., Sukhanova, A., Fleury, F., Ianoul, A., Holden, J. A., Alix, A. J., Dodson, G. G., Jardillier, J. C., Nabiev, I., and Wilkinson, A. J. (1999) Anticancer Res. 19, 317-327) suggests a pivotal role of amino acids 191-206 in catalysis. Taken together the presented data indicate that at least part(s) of the N-terminal domain regulate(s) enzyme/DNA dynamics during relaxation most probably by controlling non-covalent DNA binding downstream of the cleavage site either directly or by coordinating DNA contacts by other parts of the enzyme.

Inhibition of Flp recombinase by the topoisomerase I-targeting drugs, camptothecin and NSC-314622.

Recombinases of the lambda-Int family and type IB topoisomerases act by introducing transient single strand breaks in DNA using chemically identical reaction schemes. Recent structural data have supported the relationship between the two enzyme groups by revealing considerable similarities in the architecture of their catalytic pockets. In this study we show that the Int-type recombinase Flp is inhibited by the two structurally unrelated topoisomerase I-directed anti-cancer drugs, camptothecin (CPT) and NSC-314622. The interaction of these drugs with topoisomerase I is very specific with several single amino acid substitutions conferring drug resistance to the enzyme. Thus, the observed interaction of CPT and NSC-314622 with Flp, which is comparable to their interaction with the cleavage complex formed by topoisomerase I, strongly supports a close mechanistic and evolutionary relationship between the two enzymes. The results suggest that Flp and other Int family recombinases may provide model systems for dissecting the molecular mechanisms of topoisomerase I-directed anti-cancer therapeutic agents.

Genetic analysis of the Saccharomyces cerevisiae Sgs1 helicase defines an essential function for the Sgs1-Top3 complex in the absence of SRS2 or TOP1.

The Saccharomyces cerevisiae gene SGS1 encodes a DNA helicase that shows homology to the Escherichia coli protein RecQ and the products of the BLM and WRN genes in humans, which are defective in Bloom's and Werner's syndrome, respectively. Recently, it has been proposed that this helicase is involved in maintaining the integrity of the rDNA and that loss of Sgs1 function leads to accelerated aging. Sgs1 has been isolated on the basis of its genetic interaction with both topoisomerase I and topoisomerase III, as well as in a two-hybrid screen for proteins that interact with the C-terminal portion of topoisomerase II. We have defined the minimal structural elements of Sgs1 required for its interactions with the three topoisomerases, and demonstrate that the complex phenotypes associated with sgs1 mutants are a consequence of a dysfunctional Sgs1-Top3 complex. We also report that the synthetic relationship between mutations in SGS1 and SRS2, which encodes another helicase implicated in recombinational repair, likewise result from a dysfunctional Sgs1-Top3 interaction. Our findings indicate that Sgs1 may act on different DNA structures depending on the activity of topoisomerase I, Srs2 and topoisomerase III.

Homomeric interaction of the mouse Rad52 protein.

The Rad52 protein plays a crucial role in repairing DNA damage and homologous recombination, possibly by virtue of its ability to catalyze annealing of single-stranded DNA. In agreement with recent genetic data, we here present results based on the two-hybrid system, demonstrating that mouse Rad52p is able to form homomeric complexes. A small domain necessary and sufficient for the self-interaction is located in the conserved N-terminus of the protein. These data contribute to the important insights into the architecture of the multi-protein complex involved in recombinational DNA repair.

Communication between the ATPase and cleavage/religation domains of human topoisomerase IIalpha.

The DNA strand passage activity of eukaryotic topoisomerase II relies on a cascade of conformational changes triggered by ATP binding to the N-terminal domain of the enzyme. To investigate the interdomain communication between the ATPase and cleavage/religation domains of human topoisomerase IIalpha, we characterized a mutant enzyme that contains a deletion at the interface between the two domains, covering amino acids 350-407. The ATPase domain retained full activity with a rate of ATP hydrolysis that was severalfold higher than normal, but the ATPase activity was unaffected by DNA. The cleavage and religation activities of the enzyme were comparable with those of the wild-type enzyme both in the absence and presence of cancer chemotherapeutic agents. However, neither ATP nor a nonhydrolyzable ATP analog stimulated cleavage complex formation. Although both conserved domains retained full activity, the mutant enzyme was unable to coordinate these activities into strand passage. Our findings suggest that the normal conformational transitions occurring in the enzyme upon ATP binding are hampered or lacking in the mutant enzyme. Consistent with this hypothesis, the enzyme displayed an abnormal clamp closing activity. In summary, the region covering amino acids 350-407 in human topoisomerase IIalpha seems to be essential for correct interdomain communication and probably is involved in signaling ATP binding to the rest of the enzyme.

Position-specific effect of ribonucleotides on the cleavage activity of human topoisomerase II.

Beyond the normal DNA transactions mediated by topoisomerase II, we have recently demonstrated that the cleavage activity of the two human topoisomerase II isoforms is several-fold stimulated if a ribonucleotide rather than a deoxyribonucleotide is present at the scissile phosphodiester in one strand of the substrate. Here we show that ribonucleotides exert a position-specific effect on topoisomerase II-mediated cleavage without altering the sequence specificity of the enzyme. Ribonucleotides located within the 4 bp cleavage stagger stimulate topoisomerase II-mediated cleavage, whereas ribonucleotides located outside the stagger in general have an inhibitory effect. Results obtained from competition experiments indicate that the position-specific effect of ribonucleotides on topoisomerase II activity is caused by altered substrate interaction. When cleavage is performed with substrates containing one ribonucleotide in both strands or several ribonucleotides in one strand the effect of the individual ribonucleotides on cleavage is not additive. Finally, although topoisomerase II recognizes substrates with longer stretches of ribonucleotides, an RNA/DNA hybrid where one strand is composed entirely of RNA is not cleaved by the enzyme. The positional effect of ribonucleotides on topoisomerase II-mediated cleavage shares many similarities to the positional effect exerted by either abasic sites or base mismatches, demonstrating a general influence of DNA imperfections on topoisomerase II activity.

Using a biochemical approach to identify the primary dimerization regions in human DNA topoisomerase IIalpha.

Eukaryotic topoisomerase II is a nuclear enzyme essential for DNA metabolism and chromosome dynamics. The enzyme has a dimeric structure, and subunit dimerization is vital to the cellular functions and activities of the enzyme. Two biochemical approaches based on metal ion affinity chromatography and immunoprecipitation have been carried out to map the dimerization region(s) in human topoisomerase IIalpha. The results demonstrate that two regions spanning amino acids 1053-1069 and 1124-1143 are both essential for dimerization. The regions correspond to the interaction domains revealed in yeast topoisomerase II after crystallization of a central fragment of this enzyme, indicating that the overall C-terminal dimerization structure of eukaryotic topoisomerase II is conserved from yeast to human. Furthermore, linker insertion analysis has demonstrated that the two dimerization regions are located in a highly flexible part of the enzyme. Topoisomerase IIalpha mutant enzymes unable to dimerize via the C-terminal primary dimerization regions due to lack of one of the defined dimerization regions can still be forced to dimerize if DNA and an ATP analog are added to the reaction mixture. The result indicates that secondary interactions occur by ATP analog-mediated clamp closing when the subunits are brought together on DNA.

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.

Mapping of eukaryotic DNA topoisomerase I catalyzed cleavage without concomitant religation in the vicinity of DNA structural anomalies.

Sensitive sites for covalent trapping of eukaryotic topoisomerase I at DNA structural anomalies were mapped by a new method using purified enzyme and defined DNA substrates. To insure that the obtained topoisomerase I trapping patterns were not influenced by DNA sequence variations, a single DNA imperfection was placed centrally within a homonucleotide track. Mapping of topoisomerase I-mediated irreversible cleavage sites on homopolymeric DNA substrates containing mismatches showed trapping of the enzyme in several positions in close vicinity of the DNA imperfection, with a strong preference for the 5' junction between the duplex DNA and the base-pairing anomaly. On homopolymeric DNA substrates containing a nick, sites of topoisomerase I-mediated cleavage on the intact strand were located just opposite to the nick and from one to ten nucleotides 5' to the nick. Sites of enzyme-mediated cleavage next to a nick and an immobile single-stranded branch were located 5' to the strand interruption in distances of two to six nucleotides and two to ten nucleotides, respectively. Taken together these findings suggest that covalent trapping of topoisomerase I proceeds at positions adjacent to mismatches, nicks and single-stranded branches, where the cleavage reaction is allowed and the ensuing ligation reaction prevented. In principle, the developed interference method might be of general utility to define topoisomerase-DNA interactions relative to different types of structural anomalies.

Characterization of DNA topoisomerase II alpha/beta heterodimers in HeLa cells.

In mammalian cells, DNA topoisomerase II is the product of two distinct genes encoding the alpha and beta isoforms of the enzyme. Besides homodimeric topoisomerase IIalpha and IIbeta, we have recently shown that alpha/beta heterodimers constitute a third population of topoisomerase II in HeLa cells. We found that topoisomerase II heterodimers are not restricted to HeLa cells but exist in different mammalian cell types, and up to 25% of the total topoisomerase IIbeta population is involved in heterodimer formation. Studies of topoisomerase II phosphorylation in HeLa cells show that heterodimers are phosphorylated in vivo to a significantly lower level compared to homodimeric alpha enzymes, but in contrast to the latter neither heterodimers nor topoisomerase IIbeta homodimers coprecipitate together with a kinase activity that is able to mediate their phosphorylation. However, both enzymes can still be phosphorylated by exogenously added casein kinase II. The differential phosphorylation of topoisomerase II heterodimers suggests an alternative regulation of this topoisomerase II subclass compared to the homodimeric topoisomerase IIalpha counterparts.

The RNA-splicing factor PSF/p54 controls DNA-topoisomerase I activity by a direct interaction.

DNA-topoisomerase I has been implied in RNA splicing because it catalyzes RNA strand transfer and activates serine/arginine-rich RNA-splicing factors by phosphorylation. Here, we demonstrate a direct interaction between topoisomerase I and pyrimidine tract binding protein-associated splicing factor (PSF), a cofactor of RNA splicing, which forms heterodimers with its smaller homolog, the nuclear RNA-binding protein of 54 kDa (p54). Topoisomerase I, PSF, and p54 copurified in a 1:1:1 ratio from human A431 cell nuclear extracts. Specific binding of topoisomerase I to PSF (but not p54) was demonstrated by coimmunoprecipitation and by far Western blotting, in which renatured blots were probed with biotinylated topoisomerase I. Chemical cross-linking of pure topoisomerase I revealed monomeric, dimeric, and trimeric enzyme forms, whereas in the presence of PSF/p54 the enzyme was cross-linked into complexes larger than homotrimers. When topoisomerase I was complexed with PSF/p54 it was 16-fold more active than the pure enzyme, which could be stimulated 5- and 16-fold by the addition of recombinant PSF or native PSF/p54, respectively. A physiological role of this stimulatory mechanism seems feasible, because topoisomerase I and PSF showed a patched colocalization in A431 cell nuclei, which varied with cell cycle.

Camptothecins inhibit the utilization of hydrogen peroxide in the ligation step of topoisomerase I catalysis.

The antitumor compounds camptothecin and its derivatives topotecan and irinotecan stabilize topoisomerase I cleavage complexes by inhibiting the religation reaction of the enzyme. Previous studies, using radiolabeled camptothecin or affinity labeling reagents structurally related to camptothecin, suggest that the agent binds at the topoisomerase I-DNA interface of the cleavage complexes, interacting with both the covalently bound enzyme and with the +1 base. In this study, we have investigated the molecular mechanism of camptothecin action further by taking advantage of the ability of topoisomerase I to couple non-DNA nucleophiles to the cleaved strand of the covalent enzyme-DNA complexes. This reaction of topoisomerase I was originally observed at moderate basic pH where active cleavage complexes mediate hydrolysis or alcoholysis by accepting water or polyhydric alcohol compounds as substitutes for a 5'-OH DNA end in the ligation step. Here, we report that a H2O2-derived nucleophile, presumably, the peroxide anion, facilitates the release of topoisomerase I from the cleavage complexes at neutral pH, and we present evidence showing that this reaction is mechanistically analogous to DNA ligation. We find that camptothecin, topotecan, and SN-38 (the active metabolite of irinotecan) inhibit H2O2 ligation mediated by cleavage complexes not containing DNA downstream of the cleavage site, indicating that drug interaction with DNA 3' to the covalently bound enzyme is not strictly required for the inhibition, although the presence of double-stranded DNA in this region enhances the drug effect. The results suggest that camptothecins prevent ligation by blocking the active site of the covalently bound enzyme.

Alcoholysis and strand joining by the Flp site-specific recombinase. Mechanistically equivalent reactions mediated by distinct catalytic configurations.

The strand joining step of recombination mediated by the Flp site-specific recombinase involves the attack of a 3'-phosphotyrosyl bond by a 5'-hydroxyl group from DNA. The nucleophile in this reaction, the 5'-OH, can be substituted by glycerol or other polyhydric alcohols. The strand joining and glycerolysis reactions are mechanistically equivalent and are competitive to each other. The target diester in strand joining can be a 3'-phosphate covalently linked either to a short tyrosyl peptide or to the whole Flp protein via Tyr-343. By contrast, only the latter type of 3'-phosphotyrosyl linkage is a substrate for glycerolysis. As a result, in activated DNA substrates (containing the scissile phosphate linked to a short Flp peptide), Flp(Y343F) can mediate the joining reaction utilizing the 5'-hydroxyl attack but fails to promote glycerolysis. Wild type Flp promotes both reactions in these substrates. The strand joining and glycerolysis reactions are absolutely dependent on the catalytic histidine at position 305 of Flp. Our results fit into a model in which a Flp dimer, with one monomer covalently attached to the 3'-phosphate, is essential for orienting the target diester or the nucleophile (or both) during glycerolysis. The requirement for this dimeric complex is relaxed in the strand joining reaction because of the ability of DNA to orient the nucleophile (5'-OH) by complementary base pairing. The experimental outcomes described here have parallels to the "cleavage-dependent ligation" carried out by a catalytic variant of Flp, Flp(R308K) (Zhu, X.-D., and Sadowski, P. D. (1995) J. Biol. Chem. 270, 23044-23054).

The yeast site-specific recombinase Flp mediates alcoholysis and hydrolysis of the strand cleavage product: mimicking the strand-joining reaction with non-DNA nucleophiles.

The yeast site-specific recombinase Flp is covalently linked to DNA via a 3'-phosphotyrosyl bond during the strand-breakage step of recombination. We show that this phosphotyrosyl diester bond formed between Flp and DNA can serve as the target for alcoholysis or hydrolysis in an Flp-assisted reaction. Flp does not mediate alcoholysis of the labile phosphodiester bond within the DNA chain under our assay conditions. The body of available evidence supports the notion that the alcoholysis/hydrolysis reaction is mechanistically analogous to the strand-joining step of the recombination pathway. The only difference is that the DNA 5'-hydroxyl group that acts as the nucleophile during recombination is substituted by a non-DNA nucleophile. We find that the alcoholysis reaction occurs only within the normal cleavage complex produced by the "shared active site" assembled at the interface of two Flp monomers. Unlike the strand-joining reaction, alcoholysis does not occur on an activated DNA substrate linked at its 3'-phosphate end to a short tyrosyl peptide (not to the full-length Flp), and bound non-covalently by a Flp monomer. However, even in this substrate that mimics the strand-cleaved state, the joining reaction is competitively inhibited by a polyhydric alcohol such as glycerol.

Detection of human topoisomerase II alpha in cell lines and tissues: characterization of five novel monoclonal antibodies.

We report five novel monoclonal antibodies (Ki-S1, Ki-S4, Ki-S6, Ki-S7, and Ki-S8) reactive with a proliferation-related nuclear antigen. In immunoprecipitation and Western blot experiments using crude nuclear extracts, they recognized a protein of 170 kD that, after proteolytic digestion of the immunoprecipitate and sequencing of the resulting peptides, was identified as the alpha-isoform of human topoisomerase II. This was confirmed by testing the antibodies on a highly purified enzyme preparation. Crossreactivity with topoisomerase II beta was ruled out by testing the antibodies on crude extracts from yeast cells expressing the beta-isoform exclusively. The antibodies bind the antigen with different affinities and at different epitopes, apparently located within the carboxyl third of the enzyme. All five antibodies are suitable for archival material after adequate antigen retrieval, thereby enabling retrospective studies. This report illustrates the tissue and subcellular distribution of the antigen through the cell cycle by immunohistochemistry and confocal fluorescence microscopy. The antibodies will be useful tools in further analysis of morphological and functional aspects of topoisomerase II and may serve diagnostic purposes, as well as providing prognostic information in tumor pathology.

The effect of camptothecin on topoisomerase I catalysis.

The existence of a covalent intermediate in topoisomerase I catalysis allows uncoupling of the cleavage and ligation half-reactions on partially single-stranded DNA substrates containing a highly preferred interaction site. Using this model DNA substrate system we have demonstrated that the cleavage reaction requires bipartite interaction with two distinct DNA duplex regions; One located around the cleavage site (region A) and another located on the side holding the 5'-OH end generated by cleavage (region B). The postcleavage complexes containing the enzyme covalently attached at an internal position are capable of ligating DNA strands matching the noncleaved strand. Previously, we have characterized the effect of the antitumor agent camptothecin on the two half-reactions of topoisomerase I catalysis on DNA substrates allowing bipartite DNA interaction. The obtained results demonstrated that the drug only inhibited the ligation reaction leaving the cleavage reaction unaffected at the studied site. Here, we report that camptothecin also impairs ligation of the cleaved strand to a dinucleotide within region A in the absence of additional DNA contacts. When these results are taken together with the observation that camptothecin-trapped topoisomerase I-DNA complexes preferentially are generated at sites containing guanine immediately 3' to the cleavage position, it suggests that camptothecin inhibits the ligation reaction by forming a reversible ternary complex with the enzyme and DNA at the cleavage site within region A.

Active heterodimers are formed from human DNA topoisomerase II alpha and II beta isoforms.

DNA topoisomerase II is a nuclear enzyme essential for chromosome dynamics and DNA metabolism. In mammalian cells, two genetically and biochemically distinct topoisomerase II forms exist, which are designated topoisomerase II alpha and topoisomerase II beta. In our studies of human topoisomerase II, we have found that a substantial fraction of the enzyme exists as alpha/beta heterodimers in HeLa cells. The ability to form heterodimers was verified when human topoisomerases II alpha and II beta were coexpressed in yeast and investigated in a dimerization assay. Analysis of purified heterodimers shows that these enzymes maintain topoisomerase II specific catalytic activities. The natural existence of an active heterodimeric subclass of topoisomerase II merits attention whenever topoisomerases II alpha and II beta function, localization, and cell cycle regulation are investigated.

Analysis of functional domain organization in DNA topoisomerase II from humans and Saccharomyces cerevisiae.

The functional domain structure of human DNA topoisomerase IIalpha and Saccharomyces cerevisiae DNA topoisomerase II was studied by investigating the abilities of insertion and deletion mutant enzymes to support mitotic growth and catalyze transitions in DNA topology in vitro. Alignment of the human topoisomerase IIalpha and S. cerevisiae topoisomerase II sequences defined 13 conserved regions separated by less conserved or differently spaced sequences. The spatial tolerance of the spacer regions was addressed by insertion of linkers. The importance of the conserved regions was assessed through deletion of individual domains. We found that the exact spacing between most of the conserved domains is noncritical, as insertions in the spacer regions were tolerated with no influence on complementation ability. All conserved domains, however, are essential for sustained mitotic growth of S. cerevisiae and for enzymatic activity in vitro. A series of topoisomerase II carboxy-terminal truncations were investigated with respect to the ability to support viability, cellular localization, and enzymatic properties. The analysis showed that the divergent carboxy-terminal region of human topoisomerase IIalpha is dispensable for catalytic activity but contains elements that specifically locate the protein to the nucleus.