Interaction of Topotecan,DNA Topoisomerase I Inhibitor,with Double-stranded Polydeoxyribonucleotides. 3. Binding at the Minor Groove

Interaction of topotecan (TPT) with calf thymus DNA, coliphage T4 DNA, and poly(dGdC) · poly(dG-dC) was studied by optical (linear flow dichroism, UV-vis spectroscopy) and quantum chemical methods. The linear dichroism signal of TPT bound to DNA was shown to have positive sign in the range 260–295 nm. This means that the plane of quinoline fragment (rings A and B) of TPT forms an angle less than 54° with the long axis of DNA, and hence the TPT molecule cannot intercalate between DNA base pairs. TPT was established to bind to calf thymus DNA as readily as to coliphage T4 DNA whose cytosines in the major groove were all glycosylated at the 5th position. Consequently, the DNA major groove does not participate in TPT binding. TPT molecule was shown to compete with distamycin for binding sites in the minor groove of DNA and poly(dG-dC) · poly(dG-dC). Thus, it was demonstrated for the first time that TPT binds to DNA at its minor groove.     

Interaction of Topotecan,DNA Topoisomerase I Inhibitor,with Double-stranded Polydeoxyribonucleotides. 4. Topotecan Binds Preferably to the GC Base Pairs of DNA

Interaction of topotecan (TPT) with synthetic double-stranded polydeoxyribonucleotides has been studied in solutions of low ionic strength at pH 6.8 by linear flow dichroism (LD), circular dichroism (CD), UV-Vis absorption, and Raman spectroscopy. The complexes of TPT with poly(dG-dC)·poly(dG-dC), poly(dG)·poly(dC), poly(dA-dC)·poly(dG-dT), and poly(dA)·poly(dT), as well as complexes of TPT with calf thymus DNA and coliphage T4 DNA studied by us previously, have been shown to have negative LD in the long-wavelength absorption band of TPT, whereas the complex of TPT with poly(dA-dT)·poly(dA-dT) has positive LD in this absorption band of TPT. Thus, there are two different types of TPT complex with the polymers. TPT has been established to bind preferably to GC base pairs because its affinity to the polymers of different composition decreases in the following order: poly(dG-dC)·poly(dG-dC) > poly(dG)·poly(dC) > poly(dA-dC)·poly(dG-dT) > poly(dA)·poly(dT). The presence of DNA has been shown to shift the monomer–dimer equilibrium in TPT solutions toward dimer formation. Several duplexes of the synthetic polynucleotides bound together by bridges of TPT dimers may participate in the formation of the studied type of TPT–polynucleotide complex. Molecular models of TPT complex with linear and circular supercoiled DNAs and with deoxyguanosine have been considered. TPT (and presumably the whole camptothecin family) proved to represent a new class of DNA-specific ligands whose biological action is associated with formation of dimeric bridges between two DNA duplexes.     

Interaction of Topotecan,DNA Topoisomerase I Inhibitor,with Double-Stranded Polydeoxyribonucleotides. 5. Topotecan is Capable of Producing Single- and Double-Strand Breaks in Circular Supercoiled DNA in the Absence of the Enzyme

A study was made of the temperature, concentration, and time dependences for the emergence of breaks in the sugar-phosphate backbone of a circular supercoiled DNA (scDNA) in the presence of a campto-thecin derivative topotecan (TPT) and in the absence of DNA topoisomerase I (topo I). The experiments were carried out in low ionic strength solutions (10 mM sodium cacodylate) at neutral pH (6.8). The incubation time necessary for the appearance of double-strand breaks in scDNA in the presence of TPT correlated with the time of formation of strong TPT–DNA complex. This is the first demonstration that molecules of the camptothecin family can cause double-strand breaks in scDNA in the absence of the enzyme. A model is suggested for the complex composed of two crossed DNA duplexes bound through a bridge of two dimers of the TPT lactone form. According to this model, two carbonyl groups of D rings of different TPT dimers form hydrogen bonds with 2-amino groups of guanines located in the neighboring base pairs of different strands of one DNA duplex. At the same time, two other carbonyl groups of D rings of TPT dimers form hydrogen bonds with 2-amino groups of guanines 5 bp apart in one and the same strand of the second DNA duplex.     

Interaction of clinically important human DNA topoisomerase I poison, topotecan, with double-stranded DNA

Topotecan (TPT), a water-soluble derivative of camptothecin, is a potent antitumor poison of human DNA topoisomerase I (top1) that stabilizes the cleavage complex between the enzyme and DNA. The role of the recently discovered TPT affinity to DNA remains to be defined. The aim of this work is to clarify the molecular mechanisms of the TPT-DNA interaction and to propose the models of TPT-DNA complexes in solution in the absence of top1. It is shown that TPT molecules form dimers with a dimerization constant of (4.0 +/- 0.7) x 10(3) M(-1) and the presence of DNA provokes more than a 400-fold increase of the effective dimerization constant. Flow linear dichroism spectroscopy accompanied by circular dichroism, fluorescence, and surface-enhanced Raman scattering experiments provide evidence that TPT dimers are able to bind DNA by bridging different DNA molecules or distant DNA structural domains. This effect may provoke modification of the intrinsic geometry of the cruciform DNA structures, leading to the appearance of new crossover points that serve as the sites of the top1 loading position. The data presume the hypothesis of TPT-mediated modulation of top1-DNA recognition before ternary complex formation.     

Contribution of topoisomerase I to conversion of single-strand into double-strand DNA breaks

An in vitro system composed of nicked pBR322 DNA and purified topoisomerase I was employed to study the efficiency of the topoisomerase I-driven single-strand to double-strand DNA breaks conversion. At 1.4 × 10⁵ topoisomerase I activity units per mg DNA about 20% single-strand nicks were converted into double-strand breaks during 30 min due to topoisomerase I action. Camptothecin inhibited the conversion. The conversion was also inhibited when the relaxing activity of the used topoisomerase I was increased by phosphorylation of the enzyme with casein kinase 2. The presented data suggest that topoisomerase I may be involved in production of double-stranded breaks in irradiated cells and that this process positively depends on the amount of topoisomerase I but not on its phosphorylation state.     

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.     

DNA binding induces conformational transition within human DNA topoisomerase I in solution

We employed Raman and circular dichroism (CD) spectroscopy to probe the molecular structure of 68-kDa recombinant human DNA topoisomerase I (TopoI) in solution, in a complex with a 16-bp DNA fragment containing a camptothecin-enhanced TopoI cleavage site, and in a ternary complex with this oligonucleotide and topotecan. Raman spectroscopy reveals a TopoI secondary structure transition and significant changes in the hydrogen bonding of the tyrosine residues induced by the DNA binding. CD spectroscopy confirms the Raman data and identifies a DNA-induced (>7%) decrease of the TopoI alpha helix accompanied by at least a 6% increase of the beta structure. The Raman DNA molecular signatures demonstrated a bandshift that is expected for a net change in the environment of guanine C6 [double bond] O groups from pairing to solvent exposure. The formation of a ternary cleavage complex with TopoI, DNA, and topotecan as probed by CD spectroscopy reveals neither additional modifications of the TopoI secondary structure nor of the oligonucleotide structure, compared to the TopoI-oligonucleotide complex.     

Interleukin-2 induces the activities of DNA topoisomerase I and DNA topoisomerase II in HuT 78 cells

The induction by interleukin-2 of DNA topoisomerase I and DNA topoisomerase II activities in the human T cell line HuT 78 was investigated. HuT 78 cells were treated with 1000 U of interleukin-2/ml, and extracts of the HuT 78 nuclei were prepared over a 24 h period. The extracts were assayed quantitatively for the activities of DNA topoisomerase I and DNA topoisomerase II. Three concomitant, transient increases of 3- to 11-fold in the specific activities of both DNA topoisomerase I and DNA topoisomerase II were observed following treatment with IL-2 at 0.5, 4, and 10 h after treatment with interleukin-2. The specific activities of both enzymes returned to base-line values after each of these transient increases. These results reveal that the activities of DNA topoisomerase I and DNA topoisomerase II are highly regulated in HuT 78 cells upon treatment with IL-2.     

Effects of camptothecin, an inhibitor of DNA topoisomerase I on ribosomal gene structure and function in TG cells.

The effects of camptothecin treatment and topoisomerase I inhibition on ribosomal gene structure and function were investigated in TG cells, a human tumour cell line. 90- and 180-min treatments with 25 microM camptothecin resulted in an increased DNA fragmentation and decreased activity of topoisomerase I in cell extracts. After 180-min treatment, the incorporation of labelled uridine into total cell RNA was reduced to 39% and the ribosomal RNA synthesis to 10%, as compared to values of control cells. At the ultrastructural level, the nucleolar components appeared to be segregated; after selective DNA staining, with osmium-amine complex, a part of the nucleolar chromatin of treated cells showed the presence of thin, extended DNA filaments, superimposable to those present in control cells.     

Biological activity of bis-benzimidazole derivatives on DNA topoisomerase I and HeLa,MCF7 and A431 cells

Benzimidazoles of both natural and synthetic sources are the key components of many bio-active compounds. Several reports have shown antifungal, antiviral, H₂ receptor blocker and antitumor activities for benzimidazoles and their derivatives. In this study, we synthesized twelve bis-benzimidazole derivatives by selecting di(1H-benzo[d]imidazol-2-yl)methane as the main compound. The numbers of carbons at 2 positions of bis-benzimidazole derivatives were changed from 1 to 4, and derivatives were synthesized with methyl substitutions at 5- and/or 6- positions. The compounds were screened via in vitro plasmid superciol relaxation assays using mammalian DNA topoisomerase I and cytostatic assays were carried out against HeLa (cervix adenocarcinoma), MCF7 (breast adenocarcinoma) and A431 (skin epidermoid carcinoma) cells for selected derivatives. Our results suggest that the malonic acid derivatives of bis-benzimidazoles, namely, bis(5-methyl-1H-benzo[d]imidazol-2-yl)methane and bis(5,6-dimethyl-1H-benzo[d]imidazol-2-yl)methane, were remarkably active compounds in interfering with DNA topoisomerase I and the former compound was also found to be cytotoxic against MCF7 and A431 cells. The inhibitory effects obtained with these derivatives are significant as these compounds can be potential sources of anticancer agents.     

The Mechanism of Supercoiled DNA Recognition by Eukaryotic Type I Topoisomerases: II. A Comparison of the Enzyme Interaction with Specific and Nonspecific Oligonucleotides

Data on the interaction of DNA type I topoisomerases from the murine and human placenta cells with specific and nonspecific oligonucleotides of various structures and lengths are summarized. The relative contributions of various contacts between the enzymes and DNA that have previously been detected by X-ray analysis to the total affinity of the topoisomerases for DNA substrates are estimated. Factors that determine the differences in the enzyme interactions with specific and nonspecific single- and double-stranded DNAs are revealed. The results of the X-ray analysis of human DNA topoisomerase I are interpreted taking into account data on the comprehensive thermodynamic and kinetic analysis of the enzyme interaction with the specific and nonspecific DNAs.     

Molecular Design,Synthesis and Docking Study of Alkyl and Benzyl Derivatives of Robustic Acid as Topoisomerase I Inhibitors

Robustic acid is reported to be a bioactive compound, isolated from the medicinal plant Dalbergia benthamii Prain . Ten alkyl and benzyl derivatives ( 2a – 2j ) of robustic acid were designed and synthesized based on molecular docking approaches. The biological activities of most of the synthesized compounds (such as 2g , 2h , and 2i ) were closely consistent with the docking results. In particular, 4‐O‐phenylpropyl substituted compound 2g displayed potent topoisomerase I inhibitory activity as well as cytotoxicity against SMMC‐7721, HepG2, and HeLa cell lines. Further biological testing suggests that compound 2g acted mainly by an arrest of the tumor cells in G1 phase of the cell cycle and suppressed cell proliferation by inducing apoptosis. The findings of this study are encouraging with respect to potential utilization of these compounds as new topoisomerase I inhibitors.     

The Schrödinger''s Cat Quandary in Cell Biology: Integration of Live Cell Functional Assays with Measurements of Fixed Cells in Analysis of Apoptosis

The existing cytometric methodologies do not allow one to directly correlate, within the same cells, functional cell attributes that are revealed supravitally with features that require cell fixation to be detected or measured. Taking advantage of the "file merge" feature of the laser-scanning cytometer, we have been able to correlate the supravital changes that occur during apoptosis, namely the drop in mitochondrial transmembrane potential (Delta Psim) and generation of the reactive oxygen intermediates (ROIs), with features revealed by analysis of fixed cells: the cell cycle position and DNA fragmentation. The cell cycle position was established based on the cell's stainability with propidium iodide while DNA fragmentation was assessed by in situ DNA strand break labeling using exogenous terminal deoxynucleotidyltransferase. During apoptosis of HL-60 cells induced by the DNA topoisomerase I inhibitor camptothecin (CPT), the dissipation of Delta Psim occurred preferentially in S-phase cells and preceded the appearance of DNA strand breaks. Essentially all cells with DNA strand breaks had dissipated Delta Psim. Compared to the decrease of Delta Psim, the CPT-induced rise in ROIs during apoptosis was less restricted to S-phase cells. Furthermore, no elevation of ROIs was detected in a significant proportion of cells with DNA strand breaks. The data suggest that DNA fragmentation may occur in some cells prior to the increase in ROIs and thus, unlike the dissipation of Delta Psim, the oxidative stress may not be a prerequisite for activation of an endonuclease. Alternatively, the oxidative stress may be a transient event, occupying a narrow "time window" during the apoptotic process. The approach opens a possibility to study direct relationships, within the same cells, between cellular changes (e.g., occurring during apoptosis, mitogenesis, differentiation, etc.) detected by functional assays of live cells and changes that cannot be analyzed supravitally.     

Genome Instability in Ataxia Telangiectasia (A-T) Families: Camptothecin-Induced Damage to Replicating DNA Discriminates between Obligate A-T Heterozygotes,A-T Homozygotes and Controls

Previously we used the topoisomerase I inhibitor camptothecin (CPT), which kills mainly S-phase cells primarily by inducing double strand breaks (DSBs) in replication forks, to show that ataxia telangiectasia (A-T) fibroblasts are defective in the repair of this particular subclass of DSBs. CPT treated A-T cells reaching G2 have abnormally high levels of chromatid exchanges, viewed as prematurely condensed G2 chromosomes (G2 PCC), compared with normal cells where aberrations are mostly chromatid breaks. Here we show that A-T lymphoblastoid cells established from individuals with different mutations in the ATM gene also exhibit increased levels of chromosomal exchanges in response to CPT, indicating that the replication-associated DSBs are misrepaired in all these cells. From family studies we show that the presence of a single mutated allele in obligate A-T heterozygotes leads to intermediate levels of chromosomal exchanges in CPT-treated lymphoblastoid cells, thus providing a functional and sensitive assay to identify these individuals.     

Mapping Topoisomerase Sites in Mitochondrial DNA with a Poisonous Mitochondrial Topoisomerase I (Top1mt)

Mitochondrial topoisomerase I (Top1mt) is a type IB topoisomerase present in vertebrates and exclusively targeted to mitochondria. Top1mt relaxes mitochondrial DNA (mtDNA) supercoiling by introducing transient cleavage complexes wherein the broken DNA strand swivels around the intact strand. Top1mt cleavage complexes (Top1mtcc) can be stabilized in vitro by camptothecin (CPT). However, CPT does not trap Top1mtcc efficiently in cells and is highly cytotoxic due to nuclear Top1 targeting. To map Top1mtcc on mtDNA in vivo and to overcome the limitations of CPT, we designed two substitutions (T546A and N550H) in Top1mt to stabilize Top1mtcc. We refer to the double-mutant enzyme as Top1mt*. Using retroviral transduction and ChIP-on-chip assays with Top1mt* in Top1mt knock-out murine embryonic fibroblasts, we demonstrate that Top1mt* forms high levels of cleavage complexes preferentially in the noncoding regulatory region of mtDNA, accumulating especially at the heavy strand replication origin O_H, in the ribosomal genes (12S and 16S) and at the light strand replication origin O_L. Expression of Top1mt* also caused rapid mtDNA depletion without affecting mitochondria mass, suggesting the existence of specific mitochondrial pathways for the removal of damaged mtDNA.     

Cell division and DNA topisomerase I activity in root meristems of pea seedlings during water stress

ABSTRACT

Low water potential, generated by PEG addition to the liquid medium of hydroponically grown pea seedlings, induces a fall in moisture content in the roots, followed by the arrest of elongation. This water stress reduces the mitotic index of root meristems during the treatment and induces the appearance of a peak of mitosis at 12 hours from the beginning of recovery. This peak suggests that during water stress the cell cycle is blocked in G2 or late S phase. In a first attempt to understand the biochemical events leading to cell cycle arrest, we tested the in vitro activity of DNA topoisomerase I extracted from stressed or control root meristems. The activity of this enzyme in extracts from stressed seedlings was lower than in controls, whereas it was higher in extracts from seedlings which had recovered from water stress for a few hours. The highest specific activity was observed with seedlings at 24 hours from the start of recovery. The fact that during stress treatments and recovery there was no variation in the synthesis of a 45 kDa protein, indicated as DNA topoisomerase I, suggested that the activity of this enzyme could be posttranslationally regulated. The hypothesis that variations in the concentration of unknown endogenous regulators of the activity of this enzyme may take place during water loss or uptake in the cytosol of meristematic cells is discussed.     

人DNA 拓扑异构酶Ⅰ在毕赤酵母中的表达及发酵条件优化

为了在体外以人DNA拓扑异构酶Ⅰ(hTopoⅠ)为靶位进行抗肿瘤化合物的快速筛选,用RT-PCR法从Hela细胞中克隆了hTopoⅠ基因ORF并在毕赤酵母中首次成功表达,表达产物可分泌到发酵上清,易于制备。蛋白酶A缺陷的重组酵母(SMD-hTopoⅠ)分泌重组酶的能力比具有蛋白酶A活性的重组酵母(X33-hTopoⅠ和KM-hTopoⅠ)更高。通过发酵条件的优化,使用BMMY(pH7．25),于20℃,每隔24h补加0．5％(V/V)的甲醇和3％(V/V)的营养液,SMD-hTopoⅠ诱导72h后可表达最高的酶活力(43000u/mL),发酵上清中hTopoⅠ可达11mg/L,约占总蛋白的10％。SDS-PAGE和Westem blot分析显示,表达的hTopoⅠ为91kD蛋白,无糖基化修饰。