Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Structure and hydration of the DNA-human topoisomerase I covalent complex.

ABSTRACT: The structure and hydration of reconstituted human topoisomerase I comprising the core and the carboxyl-terminal domains in covalent complex with 22-basepair DNA duplex has been investigated by molecular dynamics simulation. The structure and the intermolecular interactions were found to be well maintained over the simulation. The complex displays a high degree of flexibility of the contact area, confirmed by the presence of numerous water-mediated protein-DNA hydrogen bonds comparable in quantity and distribution to the direct ones. The interaction between the enzyme and the solvent also provides the key for interpreting the experimental reduction of activity or affinity observed upon single residue mutation. Finally, four long lasting water molecules are observed in the proximity of the active site, one of which in the appropriate position to accept a proton from the active Tyr723.

SUBMITTER: Chillemi G

PROVIDER: S-EPMC1301528 | biostudies-other | 2001 Jul

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Publications

Structure and hydration of the DNA-human topoisomerase I covalent complex.

Chillemi G G Castrignanò T T Desideri A A

Biophysical journal 20010701 1

The structure and hydration of reconstituted human topoisomerase I comprising the core and the carboxyl-terminal domains in covalent complex with 22-basepair DNA duplex has been investigated by molecular dynamics simulation. The structure and the intermolecular interactions were found to be well maintained over the simulation. The complex displays a high degree of flexibility of the contact area, confirmed by the presence of numerous water-mediated protein-DNA hydrogen bonds comparable in quanti ...[more]

PMID: 11423431

Similar Datasets

Immunodetection of human topoisomerase I-DNA covalent complexes.

Project description:A number of established and investigational anticancer drugs slow the religation step of DNA topoisomerase I (topo I). These agents induce cytotoxicity by stabilizing topo I-DNA covalent complexes, which in turn interact with advancing replication forks or transcription complexes to generate lethal lesions. Despite the importance of topo I-DNA covalent complexes, it has been difficult to detect these lesions within intact cells and tumors. Here, we report development of a monoclonal antibody that specifically recognizes covalent topo I-DNA complexes, but not free topo I or DNA, by immunoblotting, immunofluorescence or flow cytometry. Utilizing this antibody, we demonstrate readily detectable topo I-DNA covalent complexes after treatment with camptothecins, indenoisoquinolines and cisplatin but not nucleoside analogues. Topotecan-induced topo I-DNA complexes peak at 15-30 min after drug addition and then decrease, whereas indotecan-induced complexes persist for at least 4 h. Interestingly, simultaneous staining for covalent topo I-DNA complexes, phospho-H2AX and Rad51 suggests that topotecan-induced DNA double-strand breaks occur at sites distinct from stabilized topo I-DNA covalent complexes. These studies not only provide new insight into the action of topo I-directed agents, but also illustrate a strategy that can be applied to study additional topoisomerases and their inhibitors in vitro and in vivo.

| S-EPMC4824114 | biostudies-literature

Use of double-stranded DNA mini-circles to characterize the covalent topoisomerase-DNA complex.

Project description:The enzymatic DNA relaxation requires the DNA to be transiently nicked and rejoined, the covalent topoisomerase-DNA complex being a key intermediate of the nicking-joining reaction. Practically, this reaction is most often characterized by oligonucleotides. However, the incision-religation of an oligonucleotide does not fully recapitulate the incision-religation occuring during relaxation and the preferred substrate for such reaction characterization is supercoiled DNA. We therefore developed a method that used radiolabeled supercoiled DNA mini-circles to characterize the covalent enzyme-DNA complex formed during a relaxation reaction. Resolution of the relaxation products under different conditions permitted to quantify the proportion of covalent complex formed during the relaxation catalyzed by two topoisomerase models, the Escherichia coli topoisomerase I and the calf thymus topoisomerase I. As expected, the covalent complex formed with the calf thymus topoisomerase I was significantly enriched by camptothecin, a widely-used inhibitor of this topoisomerase, and a salt jump permitted the multiple topoisomerases trapped per mini-circle to complete the reaction cycle. The identified positions of the camptothecin-induced incision sites were shown to be independent of the linking number and the substrate circular nature Overall, our results demonstrate that supercoiled mini-circles constitute a powerful and polyvalent substrate to characterize the mechanism of action of novel topoisomerases and inhibitors, including the incision-religation reaction.

| S-EPMC4547392 | biostudies-literature

Covalent Complex of DNA and Bacterial Topoisomerase: Implications in Antibacterial Drug Development.

Project description:A topoisomerase-DNA transient covalent complex can be a druggable target for novel topoisomerase poison inhibitors that represent a new class of antibacterial or anticancer drugs. Herein, we have investigated molecular features of the functionally important Escherichia coli topoisomerase I (EctopoI)-DNA covalent complex (EctopoIcc) for molecular simulations, which is very useful in the development of new antibacterial drugs. To demonstrate the usefulness of our approach, we used a model small molecule (SM), NSC76027, obtained from virtual screening. We examined the direct binding of NSC76027 to EctopoI as well as inhibition of EctopoI relaxation activity of this SM via experimental techniques. We then performed molecular dynamics (MD) simulations to investigate the dynamics and stability of EctopoIcc and EctopoI-NSC76027-DNA ternary complex. Our simulation results show that NSC76027 forms a stable ternary complex with EctopoIcc. EctopoI investigated here also serves as a model system for investigating a complex of topoisomerase and DNA in which DNA is covalently attached to the protein.

| S-EPMC7133791 | biostudies-literature

A novel norindenoisoquinoline structure reveals a common interfacial inhibitor paradigm for ternary trapping of the topoisomerase I-DNA covalent complex.

Project description:We show that five topoisomerase I inhibitors (two indenoisoquinolines, two camptothecins, and one indolocarbazole) each intercalate between the base pairs flanking the cleavage site generated during the topoisomerase I catalytic cycle and are further stabilized by a network of hydrogen bonds with topoisomerase I. The interfacial inhibition paradigm described for topoisomerase I inhibitors can be generalized to a variety of natural products that trap macromolecular complexes as they undergo catalytic conformational changes that create hotspots for drug binding. Stabilization of such conformational states results in uncompetitive inhibition and exemplifies the relevance of screening for ligands and drugs that stabilize ("trap") these macromolecular complexes.

| S-EPMC2860177 | biostudies-literature

Crystal structure of a covalent intermediate in DNA cleavage and rejoining by Escherichia coli DNA topoisomerase I.

Project description:DNA topoisomerases control DNA topology by breaking and rejoining DNA strands via covalent complexes with cleaved DNA substrate as catalytic intermediates. Here we report the structure of Escherichia coli topoisomerase I catalytic domain (residues 2-695) in covalent complex with a cleaved single-stranded oligonucleotide substrate, refined to 2.3-Å resolution. The enzyme-substrate intermediate formed after strand cleavage was captured due to the presence of the D111N mutation. This structure of the covalent topoisomerase-DNA intermediate, previously elusive for type IA topoisomerases, shows distinct conformational changes from the structure of the enzyme without bound DNA and provides detailed understanding of the covalent catalysis required for strand cleavage to take place. The portion of cleaved DNA 5' to the site of cleavage is anchored tightly with extensive noncovalent protein-DNA interactions as predicted by the "enzyme-bridged" model. Distortion of the scissile strand at the -4 position 5' to the cleavage site allows specific selectivity of a cytosine base in the binding pocket. Many antibacterial and anticancer drugs initiate cell killing by trapping the covalent complexes formed by topoisomerases. We have demonstrated in previous mutagenesis studies that accumulation of the covalent complex of bacterial topoisomerase I is bactericidal. This structure of the covalent intermediate provides the basis for the design of novel antibiotics that can trap the enzyme after formation of the covalent complex.

| S-EPMC3084087 | biostudies-literature

Protein concerted motions in the DNA-human topoisomerase I complex.

Project description:The collective motions of the core and C-terminal domains of human topoisomerase I (topo I) have been analysed by molecular dynamics simulation of the protein in covalent complex with a 22 bp DNA duplex. The analysis evidenced a great number of correlated movements of core subdomain I and II residues, and a central role for helix 5 in the protein-DNA communication, in particular with the scissile strand downstream of the cleavage site. The flow of information between these core subdomains and DNA suggests that subdomains I and II play an essential role in the DNA relaxation process. In core subdomain III the majority of DNA contacting residues do not communicate with protein regions far from DNA, suggesting that they have a structural role. However, selected core subdomain III residues, involved in the orientation of the active site region, show correlated movements with residues distant from DNA, indicating that the information concerning the catalytic event is also transmitted. The flexibility of two loops formed by residues 519-520 and 580-584 seems indispensable to the dynamic participation of core subdomain III to the DNA cleavage and religation steps. The motion of specific residues has also been found to explain the effect of single point mutations that make topo I resistant to the anticancer drug camptothecin.

| S-EPMC149835 | biostudies-literature

Crystal structure of a bacterial topoisomerase IB in complex with DNA reveals a secondary DNA binding site.

Project description:Type IB DNA topoisomerases (TopIB) are monomeric enzymes that relax supercoils by cleaving and resealing one strand of duplex DNA within a protein clamp that embraces a approximately 21 DNA segment. A longstanding conundrum concerns the capacity of TopIB enzymes to stabilize intramolecular duplex DNA crossovers and form protein-DNA synaptic filaments. Here we report a structure of Deinococcus radiodurans TopIB in complex with a 12 bp duplex DNA that demonstrates a secondary DNA binding site located on the surface of the C-terminal domain. It comprises a distinctive interface with one strand of the DNA duplex and is conserved in all TopIB enzymes. Modeling of a TopIB with both DNA sites suggests that the secondary site could account for DNA crossover binding, nucleation of DNA synapsis, and generation of a filamentous plectoneme. Mutations of the secondary site eliminate synaptic plectoneme formation without affecting DNA cleavage or supercoil relaxation.

| S-EPMC2886027 | biostudies-literature

Structure of an 'open' clamp type II topoisomerase-DNA complex provides a mechanism for DNA capture and transport.

Project description:Type II topoisomerases regulate DNA supercoiling and chromosome segregation. They act as ATP-operated clamps that capture a DNA duplex and pass it through a transient DNA break in a second DNA segment via the sequential opening and closure of ATPase-, G-DNA- and C-gates. Here, we present the first 'open clamp' structures of a 3-gate topoisomerase II-DNA complex, the seminal complex engaged in DNA recognition and capture. A high-resolution structure was solved for a (full-length ParE-ParC55)2 dimer of Streptococcus pneumoniae topoisomerase IV bound to two DNA molecules: a closed DNA gate in a B-A-B form double-helical conformation and a second B-form duplex associated with closed C-gate helices at a novel site neighbouring the catalytically important ?-pinwheel DNA-binding domain. The protein N gate is present in an 'arms-wide-open' state with the undimerized N-terminal ParE ATPase domains connected to TOPRIM domains via a flexible joint and folded back allowing ready access both for gate and transported DNA segments and cleavage-stabilizing antibacterial drugs. The structure shows the molecular conformations of all three gates at 3.7 Å, the highest resolution achieved for the full complex to date, and illuminates the mechanism of DNA capture and transport by a type II topoisomerase.

| S-EPMC3834822 | biostudies-literature

A ubiquitin-proteasome pathway for the repair of topoisomerase I-DNA covalent complexes.

Project description:Reversible topoisomerase I (Top1)-DNA cleavage complexes are the key DNA lesion induced by anticancer camptothecins (e.g. topotecan and irinotecan) as well as structurally perturbed DNAs (e.g. oxidatively damaged DNA, UV-irradiated DNA, alkylated DNA, uracil-substituted DNA, mismatched DNA, gapped and nicked DNA, and DNA with abasic sites). Top1 cleavage complexes arrest transcription and trigger transcription-dependent degradation of Top1, a phenomenon termed Top1 down-regulation. In the current study, we have investigated the role of Top1 down-regulation in the repair of Top1 cleavage complexes. Using quiescent (serum-starved) human WI-38 cells, camptothecin (CPT) was shown to induce Top1 down-regulation, which paralleled the induction of DNA single-strand breaks (SSBs) (assayed by comet assays) and ATM autophosphorylation (at Ser-1981). Interestingly, Top1 down-regulation, induction of DNA SSBs and ATM autophosphorylation were all abolished by the proteasome inhibitor MG132. Furthermore, studies using immunoprecipitation and dominant-negative ubiquitin mutants have suggested a specific requirement for the assembly of Lys-48-linked polyubiquitin chains for CPT-induced Top1 down-regulation. In contrast to the effect of proteasome inhibition, inactivation of PARP1 was shown to increase the amount of CPT-induced SSBs and the level of ATM autophosphorylation. Together, these results support a model in which Top1 cleavage complexes arrest transcription and activate a ubiquitin-proteasome pathway leading to the degradation of Top1 cleavage complexes. Degradation of Top1 cleavage complexes results in the exposure of Top1-concealed SSBs for repair through a PARP1-dependent process.

| S-EPMC2475699 | biostudies-literature

Etoposide quinone is a covalent poison of human topoisomerase IIβ.

Project description:Etoposide is a topoisomerase II poison that is utilized to treat a broad spectrum of human cancers. Despite its wide clinical use, 2-3% of patients treated with etoposide eventually develop treatment-related acute myeloid leukemias (t-AMLs) characterized by rearrangements of the MLL gene. The molecular basis underlying the development of these t-AMLs is not well understood; however, previous studies have implicated etoposide metabolites (i.e., etoposide quinone) and topoisomerase IIβ in the leukemogenic process. Although interactions between etoposide quinone and topoisomerase IIα have been characterized, the effects of the drug metabolite on the activity of human topoisomerase IIβ have not been reported. Thus, we examined the ability of etoposide quinone to poison human topoisomerase IIβ. The quinone induced ~4 times more enzyme-mediated DNA cleavage than did the parent drug. Furthermore, the potency of etoposide quinone was ~2 times greater against topoisomerase IIβ than it was against topoisomerase IIα, and the drug reacted ~2-4 times faster with the β isoform. Etoposide quinone induced a higher ratio of double- to single-stranded breaks than etoposide, and its activity was less dependent on ATP. Whereas etoposide acts as an interfacial topoisomerase II poison, etoposide quinone displayed all of the hallmarks of a covalent poison: the activity of the metabolite was abolished by reducing agents, and the compound inactivated topoisomerase IIβ when it was incubated with the enzyme prior to the addition of DNA. These results are consistent with the hypothesis that etoposide quinone contributes to etoposide-related leukemogenesis through an interaction with topoisomerase IIβ.

| S-EPMC4033626 | biostudies-other

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data