Tightening of DNA knots by supercoiling facilitates their unknotting by type II DNA topoisomerases.

Using numerical simulations, we compare properties of knotted DNA molecules that are either torsionally relaxed or supercoiled. We observe that DNA supercoiling tightens knotted portions of DNA molecules and accentuates the difference in curvature between knotted and unknotted regions. The increased curvature of knotted regions is expected to make them preferential substrates of type IIA topoisomerases because various earlier experiments have concluded that type IIA DNA topoisomerases preferentially interact with highly curved DNA regions. The supercoiling-induced tightening of DNA knots observed here shows that torsional tension in DNA may serve to expose DNA knots to the unknotting action of type IIA topoisomerases, and thus explains how these topoisomerases could maintain a low knotting equilibrium in vivo, even for long DNA molecules.

Genetic studies on the role of type IA DNA topoisomerases in DNA metabolism and genome maintenance in Escherichia coli

Le surenroulement de l’ADN est important pour tous les processus cellulaires qui requièrent la séparation des brins de l’ADN. Il est régulé par l’activité enzymatique des topoisomérases. La gyrase (gyrA et gyrB) utilise l’ATP pour introduire des supertours négatifs dans l’ADN, alors que la topoisomérase I (topA) et la topoisomérase IV (parC et parE) les éliminent. Les cellules déficientes pour la topoisomérase I sont viables si elles ont des mutations compensatoires dans un des gènes codant pour une sous-unité de la gyrase. Ces mutations réduisent le niveau de surenroulement négatif du chromosome et permettent la croissance bactérienne. Une de ces mutations engendre la production d'une gyrase thermosensible. L’activité de surenroulement de la gyrase en absence de la topoisomérase I cause l’accumulation d’ADN hyper-surenroulé négativement à cause de la formation de R-loops. La surproduction de la RNase HI (rnhA), une enzyme qui dégrade l’ARN des R-loops, permet de prévenir l’accumulation d’un excès de surenroulement négatif. En absence de RNase HI, des R-loops sont aussi formés et peuvent être utilisés pour déclencher la réplication de l’ADN indépendamment du système normal oriC/DnaA, un phénomène connu sous le nom de « constitutive stable DNA replication » (cSDR). Pour mieux comprendre le lien entre la formation de R-loops et l’excès de surenroulement négatif, nous avons construit un mutant conditionnel topA rnhA gyrB(Ts) avec l’expression inductible de la RNase HI à partir d’un plasmide. Nous avons trouvé que l’ADN des cellules de ce mutant était excessivement relâché au lieu d'être hypersurenroulé négativement en conditions de pénurie de RNase HI. La relaxation de l’ADN a été montrée comme étant indépendante de l'activité de la topoisomérase IV. Les cellules du triple mutant topA rnhA gyrB(Ts) forment de très longs filaments remplis d’ADN, montrant ainsi un défaut de ségrégation des chromosomes. La surproduction de la topoisomérase III (topB), une enzyme qui peut effectuer la décaténation de l’ADN, a corrigé les problèmes de ségrégation sans toutefois restaurer le niveau de surenroulement de l’ADN. Nous avons constaté que des extraits protéiques du mutant topA rnhA gyrB(Ts) pouvaient inhiber l’activité de surenroulement négatif de la gyrase dans des extraits d’une souche sauvage, suggérant ainsi que la pénurie de RNase HI avait déclenché une réponse cellulaire d’inhibition de cette activité de la gyrase. De plus, des expériences in vivo et in vitro ont montré qu’en absence de RNase HI, l’activité ATP-dépendante de surenroulement négatif de la gyrase était inhibée, alors que l’activité ATP-indépendante de cette enzyme demeurait intacte. Des suppresseurs extragéniques du défaut de croissance du triple mutant topA rnhA gyrB(Ts) qui corrigent également les problèmes de surenroulement et de ségrégation des chromosomes ont pour la plupart été cartographiés dans des gènes impliqués dans la réplication de l’ADN, le métabolisme des R-loops, ou la formation de fimbriae. La deuxième partie de ce projet avait pour but de comprendre les rôles des topoisomérases de type IA (topoisomérase I et topoisomérase III) dans la ségrégation et la stabilité du génome de Escherichia coli. Pour étudier ces rôles, nous avons utilisé des approches de génétique combinées avec la cytométrie en flux, l’analyse de type Western blot et la microscopie. Nous avons constaté que le phénotype Par- et les défauts de ségrégation des chromosomes d’un mutant gyrB(Ts) avaient été corrigés en inactivant topA, mais uniquement en présence du gène topB. En outre, nous avons démontré que la surproduction de la topoisomérase III pouvait corriger le phénotype Par- du mutant gyrB(Ts) sans toutefois corriger les défauts de croissance de ce dernier. La surproduction de topoisomérase IV, enzyme responsable de la décaténation des chromosomes chez E. coli, ne pouvait pas remplacer la topoisomérase III. Nos résultats suggèrent que les topoisomérases de type IA jouent un rôle important dans la ségrégation des chromosomes lorsque la gyrase est inefficace. Pour étudier le rôle des topoisomérases de type IA dans la stabilité du génome, la troisième partie du projet, nous avons utilisé des approches génétiques combinées avec des tests de « spot » et la microscopie. Nous avons constaté que les cellules déficientes en topoisomérase I avaient des défauts de ségrégation de chromosomes et de croissance liés à un excès de surenroulement négatif, et que ces défauts pouvaient être corrigés en inactivant recQ, recA ou par la surproduction de la topoisomérase III. Le suppresseur extragénique oriC15::aph isolé dans la première partie du projet pouvait également corriger ces problèmes. Les cellules déficientes en topoisomérases de type IA formaient des très longs filaments remplis d’ADN d’apparence diffuse et réparti inégalement dans la cellule. Ces phénotypes pouvaient être partiellement corrigés par la surproduction de la RNase HI ou en inactivant recA, ou encore par des suppresseurs isolés dans la première partie du projet et impliques dans le cSDR (dnaT18::aph et rne59::aph). Donc, dans E. coli, les topoisomérases de type IA jouent un rôle dans la stabilité du génome en inhibant la réplication inappropriée à partir de oriC et de R-loops, et en empêchant les défauts de ségrégation liés à la recombinaison RecA-dépendante, par leur action avec RecQ. Les travaux rapportés ici révèlent que la réplication inappropriée et dérégulée est une source majeure de l’instabilité génomique. Empêcher la réplication inappropriée permet la ségrégation des chromosomes et le maintien d’un génome stable. La RNase HI et les topoisomérases de type IA jouent un rôle majeur dans la prévention de la réplication inappropriée. La RNase HI réalise cette tâche en modulant l’activité de surenroulement ATP-dependante de la gyrase, et en empêchant la réplication à partir des R-loops. Les topoisomérases de type IA assurent le maintien de la stabilité du génome en empêchant la réplication inappropriée à partir de oriC et des R-loops et en agissant avec RecQ pour résoudre des intermédiaires de recombinaison RecA-dépendants afin de permettre la ségrégation des chromosomes.

Peptídeos derivados da toxina ParE: síntese, estrutura e estudos de inibição de DNA topoisomerases

Pós-graduação em Biotecnologia - IQ

Transcriptional functions of DNA Topoisomerases at a genome-wide scale and a single gene levels

The DNA topology is an important modifier of DNA functions. Torsional stress is generated when right handed DNA is either over- or underwound, producing structural deformations which drive or are driven by processes such as replication, transcription, recombination and repair. DNA topoisomerases are molecular machines that regulate the topological state of the DNA in the cell. These enzymes accomplish this task by either passing one strand of the DNA through a break in the opposing strand or by passing a region of the duplex from the same or a different molecule through a double-stranded cut generated in the DNA. Because of their ability to cut one or two strands of DNA they are also target for some of the most successful anticancer drugs used in standard combination therapies of human cancers. An effective anticancer drug is Camptothecin (CPT) that specifically targets DNA topoisomerase 1 (TOP 1). The research project of the present thesis has been focused on the role of human TOP 1 during transcription and on the transcriptional consequences associated with TOP 1 inhibition by CPT in human cell lines. Previous findings demonstrate that TOP 1 inhibition by CPT perturbs RNA polymerase (RNAP II) density at promoters and along transcribed genes suggesting an involvement of TOP 1 in RNAP II promoter proximal pausing site. Within the transcription cycle, promoter pausing is a fundamental step the importance of which has been well established as a means of coupling elongation to RNA maturation. By measuring nascent RNA transcripts bound to chromatin, we demonstrated that TOP 1 inhibition by CPT can enhance RNAP II escape from promoter proximal pausing site of the human Hypoxia Inducible Factor 1 (HIF-1) and c-MYC genes in a dose dependent manner. This effect is dependent from Cdk7/Cdk9 activities since it can be reversed by the kinases inhibitor DRB. Since CPT affects RNAP II by promoting the hyperphosphorylation of its Rpb1 subunit the findings suggest that TOP 1inhibition by CPT may increase the activity of Cdks which in turn phosphorylate the Rpb1 subunit of RNAP II enhancing its escape from pausing. Interestingly, the transcriptional consequences of CPT induced topological stress are wider than expected. CPT increased co-transcriptional splicing of exon1 and 2 and markedly affected alternative splicing at exon 11. Surprisingly despite its well-established transcription inhibitory activity, CPT can trigger the production of a novel long RNA (5’aHIF-1) antisense to the human HIF-1 mRNA and a known antisense RNA at the 3’ end of the gene, while decreasing mRNA levels. The effects require TOP 1 and are independent from CPT induced DNA damage. Thus, when the supercoiling imbalance promoted by CPT occurs at promoter, it may trigger deregulation of the RNAP II pausing, increased chromatin accessibility and activation/derepression of antisense transcripts in a Cdks dependent manner. A changed balance of antisense transcripts and mRNAs may regulate the activity of HIF-1 and contribute to the control of tumor progression After focusing our TOP 1 investigations at a single gene level, we have extended the study to the whole genome by developing the “Topo-Seq” approach which generates a map of genome-wide distribution of sites of TOP 1 activity sites in human cells. The preliminary data revealed that TOP 1 preferentially localizes at intragenic regions and in particular at 5’ and 3’ ends of genes. Surprisingly upon TOP 1 downregulation, which impairs protein expression by 80%, TOP 1 molecules are mostly localized around 3’ ends of genes, thus suggesting that its activity is essential at these regions and can be compensate at 5’ ends. The developed procedure is a pioneer tool for the detection of TOP 1 cleavage sites across the genome and can open the way to further investigations of the enzyme roles in different nuclear processes.

Mechanism of topology simplification by type II DNA topoisomerases

Type II DNA topoisomerases actively reduce the fractions of knotted and catenated circular DNA below thermodynamic equilibrium values. To explain this surprising finding, we designed a model in which topoisomerases introduce a sharp bend in DNA. Because the enzymes have a specific orientation relative to the bend, they act like Maxwell's demon, providing unidirectional strand passage. Quantitative analysis of the model by computer simulations proved that it can explain much of the experimental data. The required sharp DNA bend was demonstrated by a greatly increased cyclization of short DNA fragments from topoisomerase binding and by direct visualization with electron microscopy.

Functional and regulatory characteristics of eukaryotic type II DNA topoisomerase

DNA topoisomerases are ubiquitous nuclear enzymes that govern the topological interconversions of DNA by transiently breaking/rejoining the phosphodiester backbone of one (type I) or both (type II) strands of the double helix. Consistent with these functions, topoisomerases play key roles in many aspects of DNA metabolism. Type II DNA topoisomerase (topo II) is vital for various nuclear processes, including DNA replication, chromosome segregation, and maintenance of chromosome structure. Topo II expression is regulated at multiple stages, including transcriptional, posttranscriptional, and posttranslational levels, by a multitude of signaling factors. Topo II is also the cellular target for a variety of clinically relevant anti-tumor drugs. Despite significant progress in our understanding of the role of topo II in diverse nuclear processes, several important aspects of topo II function, expression, and regulation are poorly understood. We have focused this review specifically on eukaryotic DNA topoisomerase II, with an emphasis on functional and regulatory characteristics.

DNA topoisomerase I from mycobacteria - A potential drug target

DNA topoisomerases are ubiquitous group of enzymes altering the topology of DNA by concerted breakage and rejoining of the phosphodiester backbone of DNA. The enzymes are classified based on the pattern of DNA cleavage. Type IA enzymes found in all bacteria nick the DNA and attach themselves covalently to the 5' side of the nick during the first transesterification reaction. Most of the information on this group of enzymes comes from studies with E. coli topoisomerase I and III. Members of type IA group are single subunit Zn++ metalloenzymes recognizing single stranded DNA without high degree of sequence specificity during relaxation reaction of negatively super coiled DNA. So far no inhibitors are known for this group of enzymes inspite of their important role in maintaining homeostasis of DNA topology. Molecular characterization of DNA topoisomerase I from mycobacteria has revealed some of the important features of type IA enzymes hitherto unknown and provide scope for identifying novel inhibitors. The present review describes the recent developments in the area summarizing the distinctive features of mycobacterial topoisomerase I. The enzyme has several properties not shared by either type IA or 113 enzymes with respect to DNA binding, recognition, sequence specificity and interaction pattern. The physiological basis of the unusual features is discussed. The unique properties described would aid in developing the enzyme as a target molecule in pharmaceutical design. In addition, the findings lead to address some fundamental questions on the intracellular role of topoisomerase I in the biology of mycobacteria which are one of the most formidable group of pathogenic organisms.

Inhibition of type IA topoisomerase by a monoclonal antibody through perturbation of DNA cleavage-religation equilibrium

Type IA DNA topoisomerases, typically found in bacteria, are essential enzymes that catalyse the DNA relaxation of negative supercoils. DNA gyrase is the only type II topoisomerase that can carry out the opposite reaction (i.e. the introduction of the DNA supercoils). A number of diverse molecules target DNA gyrase. However, inhibitors that arrest the activity of bacterial topoisomerase I at low concentrations remain to be identified. Towards this end, as a proof of principle, monoclonal antibodies that inhibit Mycobacterium smegmatis topoisomerase I have been characterized and the specific inhibition of Mycobacterium smegmatis topoisomerase I by a monoclonal antibody, 2F3G4, at a nanomolar concentration is described. The enzyme-bound monoclonal antibody stimulated the first transesterification reaction leading to enhanced DNA cleavage, without significantly altering the religation activity of the enzyme. The stimulated DNA cleavage resulted in perturbation of the cleavagereligation equilibrium, increasing single-strand nicks and proteinDNA covalent adducts. Monoclonal antibodies with such a mechanism of inhibition can serve as invaluable tools for probing the structure and mechanism of the enzyme, as well as in the design of novel inhibitors that arrest enzyme activity.

Characterization of DNA topoisomerase I from Mycobacterium tuberculosis: DNA cleavage and religation properties and inhibition of its activity

Type I DNA topoisomerases from bacteria catalyse relaxation of negatively supercoiled DNA in a Mg2+ dependent manner. Although topoisomerases of distinct classes have been subjected for anti-cancer and anti-infective drug development, bacterial type I enzymes are way behind in this regard. Our studies with Mycobacterium smegmatis topoisomerase I (MstopoI) revealed several of its distinct properties compared to the well studied Escherichia coli topoisomerase I (EctopoI) suggesting the possibility of targeting the mycobacterial enzyme for inhibitor development. Here, we describe Mycobacterium tuberculosis topoisomerase I (MttopoI) and compare its properties with MstopoI and EctopoI. The enzyme cleaves DNA at preferred sites in a pattern similar to its ortholog from M. smegmatis. Oligonucleotides containing the specific recognition sequence inhibited the activity of the enzyme in a manner similar to that of MstopoI. Substitution of the acidic residues, D111 and E115 which are involved in Mg2+ co-ordination, to alanines affected the DNA relaxation activity. Unlike the wild type enzyme, D111A was dependent on Mg2+ for DNA cleavage and both the mutants were compromised in religation. The monoclonal antibody (mAb), 2F3G4, developed against MstopoI inhibited the relaxation activity of MttopoI. These studies affirm the characteristics of MttopoI to be similar to MstopoI and set a stage to target it for the development of specific small molecule inhibitors. (C) 2012 Elsevier Inc. All rights reserved.

Reduction in DNA topoisomerase I level affects growth, phenotype and nucleoid architecture of Mycobacterium smegmatis

The steady-state negative supercoiling of eubacterial genomes is maintained by the action of DNA topoisomerases. Topoisomerase distribution varies in different species of mycobacteria. While Mycobacterium tuberculosis (Mtb) contains a single type I (Topol) and a single type II (Gyrase) enzyme, Mycobacterium smegmatis (Msm) and other members harbour additional relaxases. Topol is essential for Mtb survival. However, the necessity of Topol or other relaxases in Msm has not been investigated. To recognize the importance of Topol for growth, physiology and gene expression of Msm, we have developed a conditional knock-down strain of Topol in Msm. The Topol-depleted strain exhibited extremely slow growth and drastic changes in phenotypic characteristics. The cessation of growth indicates the essential requirement of the enzyme for the organism in spite of having additional DNA relaxation enzymes in the cell. Notably, the imbalance in Topol level led to the altered expression of topology modulatory proteins, resulting in a diffused nucleoid architecture. Proteomic and transcript analysis of the mutant indicated reduced expression of the genes involved in central metabolic pathways and core DNA transaction processes. RNA polymerase (RNAP) distribution on the transcription units was affected in the Topol-depleted cells, suggesting global alteration in transcription. The study thus highlights the essential requirement of Topol in the maintenance of cellular phenotype, growth characteristics and gene expression in mycobacteria. A decrease in Topol level led to altered RNAP occupancy and impaired transcription elongation, causing severe downstream effects.

DNA拓扑异构酶Ⅱ与ⅠA的进化分析

DNA拓扑异构酶与断开DNA链和改变其拓扑结构有关。先前的研究认为,拓扑异构酶TypeⅡB是由TypeⅡA通过基因的复制、重组和缺失所造成。然而,本研究结果则显示,TypeⅠA与TypeⅡB的进化关系较近,而TypeⅡA与TypeⅡB的关系较远。因此,TypeⅡB可能是由TypeⅠA演化而来,或者说,TypeⅡB是TypeⅠA在古细菌中的特化形式;TypeⅡB可能是由TypeⅡA通过形成TypeⅠA后演化而成的,而不是由TypeⅡA直接通过基因的复制、重组和缺失所造成。在由TypeⅡA演化成为TypeⅠA,最后演化为TypeⅡB的过程中,DNA拓扑异构酶的催化机制也发生了变化从需要金属离子的协助,演化到了不需要镁离子的存在协助其催化。

Identification, characteristic and phylogenetic analysis of type II DNA topoisomerase gene in Giardia lamblia

The genes encoding type II DNA topoisomerases were investigated in Giardia lamblia genome, and a type IIA gene, GlTop 2 was identified. It is a single copy gene with a 4476 by long ORF without intron. The deduced amino acid sequence shows strong homology to eukaryotic DNA Top 2. However, some distortions were found, such as six insertions in the ATPase domain and the central domain, a similar to 100 as longer central domain; a similar to 200 as shorter C-terminal domain containing rich charged residues. These features revealed by comparing with Top 2 of the host, human, might be helpful in exploiting drug selectivity for antigiardial therapy. Phylogenetic analysis of eukaryotic enzymes showed that kinetoplastids, plants, fungi, and animals were monophyletic groups, and the animal and fungi lineages shared a more recent common ancestor than either did with the plant lineage; microsporidia grouped with fungi. However, unlike many previous phylogenetic analyses, the "amitochondriate" G. lamblia was not the earliest branch but diverged after mitochondriate kinetoplastids in our trees. Both the finding of typical eukaryotic type IIA topoisomerase and the phylogenetic analysis suggest G. lamblia is not possibly as primitive as was regarded before and might diverge after the acquisition of mitochondria. This is consistent with the recent discovery of mitochondrial remnant organelles in G. lamblia.

Design and synthesis of peptides from bacterial ParE toxin as inhibitors of topoisomerases

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Two short peptides including segments of subunit A of Escherichia coli DNA gyrase as potential probes to evaluate the antibacterial activity of quinolones

Quinolones constitute a family of compounds with a potent antibiotic activity. The enzyme DNA gyrase, responsible for the replication and transcription processes in DNA of bacteria, is involved in the mechanism of action of these drugs. In this sense, it is believed that quinolones stabilize the so-called 'cleavable complex' formed by DNA and gyrase, but the whole process is still far from being understood at the molecular level. This information is crucial in order to design new biological active products. As an approach to the problem, we have designed and synthesized low molecular weight peptide mimics of DNA gyrase. These peptides correspond to sequences of the subunit A of the enzyme from Escherichia coli, that include the quinolone resistance-determining region (positions 75-92) and a segment containing the catalytic Tyr-122 (positions 116-130). The peptide mimic of the non-mutated enzyme binds to ciprofloxin (CFX) only when DNA and Mg2+ were present (Kd = 1.6 × 10 -6 m), a result previously found with DNA gyrase. On the other hand, binding was reduced when mutations of Ser-83 to Leu-83 and Asp-87 to Asn-87 were introduced, a double change previously found in the subunit A of DNA gyrase from several CFX-resistant clinical isolates of E. coli. These results suggest that synthetic peptides designed in a similar way to that described here can be used as mimics of gyrases (topoisomerases) in order to study the binding of the quinolone to the enzyme-DNA complex as well as the mechanism of action of these antibiotics. Copyright © 2001 European Peptide Society and John Wiley & Sons, Ltd.