RecQ helicase stimulates both DNA catenation and changes in DNA topology by topoisomerase III

Together, RecQ helicase and topoisomerase III (Topo III) of Escherichia coli comprise a potent DNA strand passage activity that can catenate covalently closed DNA (Harmon, F. G., DiGate, R. J., and Kowalczykowski, S. C. (1999) Mol. Cell 3, 611-620). Here we directly assessed the structure of the catenated DNA species formed by RecQ helicase and Topo III using atomic force microscopy. The images show complex catenated DNA species involving crossovers between multiple double-stranded DNA molecules that are consistent with full catenanes. E. coli single-stranded DNA-binding protein significantly stimulated both the topoisomerase activity of Topo III alone and the DNA strand passage activity of RecQ helicase and Topo III. Titration data suggest that an intermediate of the RecQ helicase unwinding process, perhaps a RecQ helicase-DNA fork, is the target for Topo III action. Catenated DNA is the predominant product under conditions of molecular crowding; however, we also discovered that RecQ helicase and single-stranded DNA-binding protein greatly stimulated the intramolecular strand passage ("supercoiling") activity of Topo III, as revealed by changes in the linking number of uncatenated DNA. Together our results demonstrate that RecQ helicase and Topo III function together to comprise a potent and concerted single-strand DNA passage activity that can mediate both catenation-decatenation processes and changes in DNA topology.     

The unique DNA topology and DNA topoisomerases of hyperthermophilic archaea

Abstract: Hyperthermophilic archaea exhibit a unique pattern of DNA topoisomerase activities. They have a peculiar enzyme, reverse gyrase, which introduces positive superturns into DNA at the expense of ATP. This enzyme has been found in all hyperthermophiles tested so far (including Bacteria) but never in mesophiles. Reverse gyrases are formed by the association of a helicase-like domain and a 5'-type I DNA topoisomerase. These two domains might be located on the same polypeptide. However, in the methanogenic archaeon Methanopyrus kandleri , the topoisomerase domain is divided between two subunits. Besides reverse gyrase, Archaea contain other type I DNA topoisomerases; in particular, M. kandleri harbors the only known procaryotic 3'-type I DNA topoisomerase (Topo V). Hyperthermophilic archaea also exhibit specific type II DNA topoisomerases (Topo II), i.e. whereas mesophilic Bacteria have a Topo II that produces negative supercoiling (DNA gyrase), the Topo II from Sulfolobus and Pyrococcus lack gyrase activity and are the smallest enzymes of this type known so far. This peculiar pattern of DNA topoisomerases in hyperthermophilic archaea is paralleled by a unique DNA topology, i.e. whereas DNA isolated from Bacteria and Eucarya is negatively supercoiled, plasmidic DNA from hyperthermophilic archaea are from relaxed to positively supercoiled. The possible evolutionary implications of these findings are discussed in this review. We speculate that gyrase activity in mesophiles and reverse gyrase activity in hyperthermophiles might have originated in the course of procaryote evolution to balance the effect of temperature changes on DNA structure.     

DNA binding properties of 9-substituted harmine derivatives

The beta-carboline alkaloids have been characterized as a group of potential antitumor agents. The underlying mechanisms of harmine and its derivatives were investigated by DNA binding assay and Topoisomerase (Topo) inhibition assay. Meanwhile, the DNA photocleavage potential of these compounds and their cytotoxicity were also examined by DNA photocleavage assay and cytotoxicity assay in vitro. Harmine and its derivatives exhibited remarkable DNA intercalation capacity and significant Topo I inhibition activity but no effect with Topo II. Introducing an appropriate substituent into position-9 of beta-carboline nucleus enhanced the affinity of the drug to DNA resulting in remarkable Topo I inhibition effects. These results suggested that the ability of these compounds to act as intercalating agents and Topo I inhibitors was related to the antitumor activity. Moreover, these data showing a correlation between cytotoxicity and Topo I inhibition or DNA binding capacity are very important as they strongly suggested that the Topo I-mediated DNA cleavage assay and DNA binding assay could be used as a guide to design and develop superior analogues for antitumor activities.     

The role of the DNA hypermethylating agent Budesonide in the decatenating activity of DNA topoisomerase II

Catenations between sister chromatids result from DNA replication and must be resolved to ensure proper chromatid segregation in mitosis. Functionally active Topoisomerase II (Topo II), through its mechanism of concerted breaking and rejoining of double stranded DNA, is required to carry out this fundamental process. In previous studies we have shown that modifications in DNA sequence by halogenated pyrimidines and by the demethylating agent 5-azacytidine leads to malfunction of Topo II that results in an increased yield of endorreduplicated cells as a result of segregation failure. In the present work we have evaluated the possible influence of the methylating agent Budesonide to modify the frequency of endoreduplicated cells in AA8 Chinese hamster cell population. Our results seem to indicate that when Budesonide was administered for two consecutive cell cycles did induce an increase in the yield of endoreduplicated cells, as previously observed for the hypomethylating agent 5-azaC. We have also examined the possible relationship between extensive hypermethylation induced by Budesonide in DNA and stabilization of cleavable complexes by m-AMSA. Taken as a whole, our results show that the degree of methylation in DNA correlates with the effectiveness of m-AMSA to stabilize the Topo II-DNA complexes and to induce DNA cleavage. These findings evidence for the first time the functional importance of DNA hyper- and hypomethylation changes as epigenetic factors able to modulate Topo II activity for proper chromosome segregation.     

Direct Monitoring of the Strand Passage Reaction of DNA Topoisomerase II Triggers Checkpoint Activation

By necessity, the ancient activity of type II topoisomerases co-evolved with the double-helical structure of DNA, at least in organisms with circular genomes. In humans, the strand passage reaction of DNA topoisomerase II (Topo II) is the target of several major classes of cancer drugs which both poison Topo II and activate cell cycle checkpoint controls. It is important to know the cellular effects of molecules that target Topo II, but the mechanisms of checkpoint activation that respond to Topo II dysfunction are not well understood. Here, we provide evidence that a checkpoint mechanism monitors the strand passage reaction of Topo II. In contrast, cells do not become checkpoint arrested in the presence of the aberrant DNA topologies, such as hyper-catenation, that arise in the absence of Topo II activity. An overall reduction in Topo II activity (i.e. slow strand passage cycles) does not activate the checkpoint, but specific defects in the T-segment transit step of the strand passage reaction do induce a cell cycle delay. Furthermore, the cell cycle delay depends on the divergent and catalytically inert C-terminal region of Topo II, indicating that transmission of a checkpoint signal may occur via the C-terminus. Other, well characterized, mitotic checkpoints detect DNA lesions or monitor unattached kinetochores; these defects arise via failures in a variety of cell processes. In contrast, we have described the first example of a distinct category of checkpoint mechanism that monitors the catalytic cycle of a single specific enzyme in order to determine when chromosome segregation can proceed faithfully.     

Computer Analysis of Conformational and Physicochemical Properties of Nucleotide Sequences Cleavable by DNA Topoisomerase I

DNA binding with enzymes is followed by specific adaptation of the DNA structure, including partial or almost complete melting, structural changes in the sugar-phosphate backbone, stretching, compressing, bending or kinking, base flipping, etc. The set of conformational changes is individual for each enzyme and is aimed at efficiently adjusting the orbitals of the reacting groups of the enzyme and the specific DNA site to 10°–15°. The efficiency of nucleotide sequence adaptation determined by the enzyme depends on several structural characteristics. Optimal adjustment is achieved only in the case of specific DNA sequences; as a result, the reaction rate is four to eight orders of magnitude higher with specific than with nonspecific sequences. DNA topoisomerase I (Topo) is a sequence-dependent enzyme. Although less efficiently, Topo cleaves sequences which differ considerably from the optimal sequence. A method based on the analysis of conformational and physicochemical properties of the DNA helix was used to examine many nucleotide sequences cleavable by Topo. The method yields detailed information on similarity or difference of DNA structural units. The cleavable sequences proved to be similar in roll, slide, twist, and rise. In addition, all sequences had sterically disadvantageous contacts between N3 and NH₂ of guanines and N3 of adenines in the minor groove, which corresponded to the presence of dinucleotides Py-Pu in the cleavage site. DNA bending towards the major groove is easier in the case of the optimal sequence. This method is promising for analyzing the efficiency of nucleic acid cleavage by various DNA- and RNA-dependent enzymes.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 3, 2005, pp. 488–496.Original Russian Text Copyright © 2005 by Oshchepkov, Bugreev, Kolchanov, Nevinsky.     

Topoisomerase I-mediated integration of hepadnavirus DNA in vitro.

Hepadnaviruses integrate in cellular DNA via an illegitimate recombination mechanism, and clonally propagated integrations are present in most hepatocellular carcinomas which arise in hepadnavirus carriers. Although integration is not specific for any viral or cellular sequence, highly preferred integration sites have been identified near the DR1 and DR2 sequences and in the cohesive overlap region of virion DNA. We have mapped a set of preferred topoisomerase I (Topo I) cleavage sites in the region of DR1 on plus-strand DNA and in the cohesive overlap near DR2 and have tested whether Topo I is capable of mediating illegitimate recombination of woodchuck hepatitis virus (WHV) DNA with cellular DNA by developing an in vitro assay for Topo I-mediated linking. Four in vitro-generated virus-cell hybrid molecules have been cloned, and sequence analysis demonstrated that Topo I can mediate both linkage of WHV DNA to 5'OH acceptor ends of heterologous DNA fragments and linkage of WHV DNA into internal sites of a linear double-stranded cellular DNA. The in vitro integrations occurred at preferred Topo I cleavage sites in WHV DNA adjacent to the DR1 and were nearly identical to a subset of integrations cloned from hepatocellular carcinomas. The end specificity and polarity of viral sequences in the integrations allows us to propose a prototype integration mechanism for both ends of a linearized hepadnavirus DNA molecule.     

Inhibition of archaeal growth and DNA topoisomerase VI activities by the Hsp90 inhibitor radicicol

Type II DNA topoisomerases have been classified into two families, Topo IIA and Topo IIB, based on structural and mechanistic dissimilarities. Topo IIA is the target of many important antibiotics and antitumoural drugs, most of them being inactive on Topo IIB. The effects and mode of action of Topo IIA inhibitors in vitro and in vivo have been extensively studied for the last twenty-five years. In contrast, studies of Topo IIB inhibitors were lacking. To document this field, we have studied two Hsp90 inhibitors (radicicol and geldanamycin), known to interact with the ATP-binding site of Hsp90 (the Bergerat fold), which is also present in Topo IIB. Here, we report that radicicol inhibits the decatenation and relaxation activities of Sulfolobus shibatae DNA topoisomerase VI (a Topo IIB) while geldanamycin does not. In addition, radicicol has no effect on the Topo IIA Escherichia coli DNA gyrase. In agreement with their different effects on DNA topoisomerase VI, we found that radicicol can theoretically fit in the ATP-binding pocket of the DNA topoisomerase VI 'Bergerat fold', whereas geldanamycin cannot. Radicicol inhibited growths of Sulfolobus acidocaldarius (a crenarchaeon) and of Haloferax volcanii (a euryarchaeon) at the same doses that inhibited DNA topoisomerase VI in vitro. In contrast, the bacteria E.coli was resistant to this drug. Radicicol thus appears to be a very promising compound to study the mechanism of Topo IIB in vitro, as well as the biological roles of these enzymes in vivo.     

Phylogenomics of type II DNA topoisomerases

Type II DNA topoisomerases (Topo II) are essential enzymes implicated in key nuclear processes. The recent discovery of a novel kind of Topo II (DNA topoisomerase VI) in Archaea led to a division of these enzymes into two non-homologous families, (Topo IIA and Topo IIB) and to the identification of the eukaryotic protein that initiates meiotic recombination, Spo11. In the present report, we have updated the distribution of all Topo II in the three domains of life by a phylogenomic approach. Both families exhibit an atypical distribution by comparison with other informational proteins, with predominance of Topo IIA in Bacteria, Eukarya and viruses, and Topo IIB in Archaea. However, plants and some Archaea contain Topo II from both families. We confront this atypical distribution with current hypotheses on the evolution of the three domains of life and origin of DNA genomes.     

Development of DNA delivery system using Pseudomonas exotoxin A and a DNA binding region of human DNA topoisomerase I

Gene therapy is defined as the delivery of a functional gene for expression in somatic tissues with the intent to cure a disease. Thus, highly efficient gene transfer is essential for gene therapy. Receptor-mediated gene delivery can offer high efficiency in gene transfer, but several technical difficulties need to be solved. In this study, we first examined the DNA binding regions of the human DNA topoisomerase I (Topo I), using agarose gel mobility shift assay, in order to identify sites of noncovalent binding of human DNA Topo I to plasmid DNA. We identified four DNA binding regions in human DNA Topo I. They resided in aa 51–200, 271–375, 422–596, and 651–696 of the human DNA Topo I. We then used one of the four regions as a DNA binding protein fragment in the construction of a DNA delivery vehicle. Based on the known functional property of each Pseudomonas exotoxin A (PE) domain and human DNA Topo I, we fused the receptor binding and membrane translocation domains of PE with a highly positively charged DNA binding region of the N-terminal 198 amino acid residues of human DNA Topo I. The resulting recombinant protein was examined for DNA binding in vitro and transfer efficiency in cultured cells. The results show that this DNA delivery protein is a general DNA delivery vehicle without DNA sequence, topology, and cell-type specificity. The DNA delivery protein could be used to target genes of interest into cells for genetic and biochemical studies. Therefore, this technique can potentially be applied to cancer gene therapy. Received: 19 July 1999 / Received revision: 10 September 1999 / Accepted: 24 September 1999     

Topoisomerase IV bends and overtwists DNA upon binding

Escherichia coli topoisomerase IV (Topo IV) is an essential ATP-dependent enzyme that unlinks sister chromosomes during replication and efficiently removes positive but not negative supercoils. In this article, we investigate the binding properties of Topo IV onto DNA in the absence of ATP using a single molecule micromanipulation setup. We find that the enzyme binds cooperatively (Hill coefficient alpha approximately 4) with supercoiled DNA, suggesting that the Topo IV subunits assemble upon binding onto DNA. It interacts preferentially with (+) rather than (-) supercoiled DNA (Kd+=0.15 nM, Kd-=0.23 nM) and more than two orders-of-magnitude more weakly with relaxed DNA (Kd0 approximately 36 nM). Like gyrase but unlike the eukaryotic Topo II, Topo IV bends DNA with a radius 0= 6.4 nm and locally changes its twist and/or its writhe by 0.16 turn per bound complex. We estimate its free energy of binding and study the dynamics of interaction of Topo IV with DNA at the binding threshold. We find that the protein/DNA complex alternates between two states: a weakly bound state where it stays with probability p = 0.89 and a strongly bound state (with probability p = 0.11). The methodology introduced here to characterize the Topo IV/DNA complex is very general and could be used to study other DNA/protein complexes.     

Multifaceted role of the Topo IIIα–RMI1-RMI2 complex and DNA2 in the BLM-dependent pathway of DNA break end resection

BLM, a RecQ family DNA helicase mutated in Bloom''s Syndrome, participates in homologous recombination at two stages: 5′ DNA end resection and double Holliday junction dissolution. BLM exists in a complex with Topo IIIα, RMI1 and RMI2. Herein, we address the role of Topo IIIα and RMI1-RMI2 in resection using a reconstituted system with purified human proteins. We show that Topo IIIα stimulates DNA unwinding by BLM in a manner that is potentiated by RMI1-RMI2, and that the processivity of resection is reliant on the Topo IIIα–RMI1-RMI2 complex. Topo IIIα localizes to the ends of double-strand breaks, thus implicating it in the recruitment of resection factors. While the single-stranded DNA binding protein RPA plays a major role in imposing the 5′ to 3′ polarity of resection, Topo IIIα also makes a contribution in this regard. Moreover, we show that DNA2 stimulates the helicase activity of BLM. Our results thus uncover a multifaceted role of the Topo IIIα–RMI1-RMI2 ensemble and of DNA2 in the DNA resection reaction.     

The domain organization and properties of individual domains of DNA topoisomerase V, a type 1B topoisomerase with DNA repair activities.

Topoisomerase V (Topo V) is a type IB (eukaryotic-like) DNA topoisomerase. It was discovered in the hyperthermophilic prokaryote Methanopyrus kandleri and is the only topoisomerase with associated apurinic/apyrimidinic (AP) site-processing activities. The structure of Topo V in the free and DNA-bound states was probed by limited proteolysis at 37 degrees C and 80 degrees C. The Topo V protein is comprised of (i) a 44-kDa NH(2)-terminal core subdomain, which contains the active site tyrosine residue for topoisomerase activity, (ii) an immediately adjacent 16-kDa subdomain that contains degenerate helix-hairpin-helix (HhH) motifs, (iii) a protease-sensitive 18-kDa HhH "hinge" region, and (iv) a 34-kDa COOH-terminal HhH domain. Three truncated Topo V polypeptides comprising the NH(2)-terminal 44-kDa and 16-kDa domains (Topo61), the 44-, 16-, and 18-kDa domains (Topo78), and the COOH-terminal 34-kDa domain (Topo34) were cloned, purified, and characterized. Both Topo61 and Topo78 are active topoisomerases, but in contrast to Topo V these enzymes are inhibited by high salt concentrations. Topo34 has strong DNA-binding ability but shows no topoisomerase activity. Finally, we demonstrate that Topo78 and Topo34 possess AP lyase activities that are important in base excision DNA repair. Thus, Topo V has at least two active sites capable of processing AP DNA. The significance of multiple HhH motifs for the Topo V processivity is discussed.     

DNA chirality-dependent stimulation of topoisomerase IV activity by the C-terminal AAA+ domain of FtsK

We have studied the stimulation of topoisomerase IV (Topo IV) by the C-terminal AAA+ domain of FtsK. These two proteins combine to assure proper chromosome segregation in the cell. Stimulation of Topo IV activity was dependent on the chirality of the DNA substrate: FtsK stimulated decatenation of catenated DNA and relaxation of positively supercoiled [(+)ve sc] DNA, but inhibited relaxation of negatively supercoiled [(−)ve sc] DNA. The DNA translocation activity of FtsK was not required for stimulation, but was required for inhibition. DNA chirality did not affect any of the activities of FtsK, suggesting that FtsK possesses an inherent Topo IV stimulatory activity that is presumably mediated by protein–protein interactions, the stability of Topo IV on the DNA substrate dictated the effect observed. Inhibition occurs because FtsK can strip distributively acting topoisomerase off (−)ve scDNA, but not from either (+)ve scDNA or catenated DNA where the enzyme acts processively. Our analyses suggest that FtsK increases the efficiency of trapping of the transfer segment of DNA during the catalytic cycle of the topoisomerase.     

Bacterial topoisomerase I and topoisomerase III relax supercoiled DNA via distinct pathways

Escherichia coli topoisomerases I and III (Topo I and Topo III) relax negatively supercoiled DNA and also catenate/decatenate DNA molecules containing single-stranded DNA regions. Although these enzymes share the same mechanism of action and have similar structures, they participate in different cellular processes. In bulk experiments Topo I is more efficient at DNA relaxation, whereas Topo III is more efficient at catenation/decatenation, probably reflecting their differing cellular roles. To examine the differences in the mechanism of these two related type IA topoisomerases, single-molecule relaxation studies were conducted on several DNA substrates: negatively supercoiled DNA, positively supercoiled DNA with a mismatch and positively supercoiled DNA with a bulge. The experiments show differences in the way the two proteins work at the single-molecule level, while also recovering observations from the bulk experiments. Overall, Topo III relaxes DNA efficiently in fast processive runs, but with long pauses before relaxation runs, whereas Topo I relaxes DNA in slow processive runs but with short pauses before runs. The combination of these properties results in Topo I having an overall faster total relaxation rate, even though the relaxation rate during a run for Topo III is much faster.     

Distinct autoreactive T cell responses to native and fragmented DNA topoisomerase I: influence of APC type and IL-2

Systemic sclerosis (SSc) is an autoimmune connective tissue disease of unknown etiology in which T cell responses to various autoantigens, including DNA topoisomerase I (Topo I), have been implicated. We investigated whether dendritic cells, generally considered to be the most potent APCs for the initiation of immune responses, would present either of two forms of Topo I to T cells more efficiently than PBMC APCS: Using cells from healthy controls and SSc patients, several important observations were made. First, neither APC type was able to initiate T cell proliferative responses to full-length native Topo I unless exogenous IL-2 was added. This is in contrast to vigorous T cell proliferation in response to Topo I polypeptide fragments presented by either APC type. Second, T cell responses to the full-length form of Topo I presented by dendritic cells were considerably lower than responses to Ag presented by PBMC APCS: Finally, no secondary T cell responses were observed unless the same Ag/APC combination as that used in the primary stimulation was maintained. These data indicate that different peptides are generated based upon the form of the Topo I and the APC that processes it. Taken together, these results suggest that a very specific combination of antigenic form and APC may be involved in breaking tolerance to Topo I in the early stages of development of SSC:     

How topoisomerase IV can efficiently unknot and decatenate negatively supercoiled DNA molecules without causing their torsional relaxation

Freshly replicated DNA molecules initially form multiply interlinked right-handed catenanes. In bacteria, these catenated molecules become supercoiled by DNA gyrase before they undergo a complete decatenation by topoisomerase IV (Topo IV). Topo IV is also involved in the unknotting of supercoiled DNA molecules. Using Metropolis Monte Carlo simulations, we investigate the shapes of supercoiled DNA molecules that are either knotted or catenated. We are especially interested in understanding how Topo IV can unknot right-handed knots and decatenate right-handed catenanes without acting on right-handed plectonemes in negatively supercoiled DNA molecules. To this end, we investigate how the topological consequences of intersegmental passages depend on the geometry of the DNA-DNA juxtapositions at which these passages occur. We observe that there are interesting differences between the geometries of DNA-DNA juxtapositions in the interwound portions and in the knotted or catenated portions of the studied molecules. In particular, in negatively supercoiled, multiply interlinked, right-handed catenanes, we detect specific regions where DNA segments belonging to two freshly replicated sister DNA molecules form left-handed crossings. We propose that, due to its geometrical preference to act on left-handed crossings, Topo IV can specifically unknot supercoiled DNA, as well as decatenate postreplicative catenanes, without causing their torsional relaxation.