RESUMO
Topoisomerases are proven drug targets, but antibiotics that poison bacterial Topoisomerase 1 (Top1) have yet to be discovered. We have developed a rapid and direct assay for quantification of Top1-DNA adducts that is suitable for high throughput assays. Adducts are recovered by "RADAR fractionation", a quick, convenient approach in which cells are lysed in chaotropic salts and detergent and nucleic acids and covalently bound adducts then precipitated with alcohol. Here we show that RADAR fractionation followed by ELISA immunodetection can quantify adducts formed by wild-type and mutant Top1 derivatives encoded by two different bacterial pathogens, Y. pestis and M. tuberculosis, expressed in E. coli or M. smegmatis, respectively. For both enzymes, quantification of adducts by RADAR/ELISA produces results comparable to the more cumbersome classical approach of CsCl density gradient fractionation. The experiments reported here establish that RADAR/ELISA assay offers a simple way to characterize Top1 mutants and analyze kinetics of adduct formation and repair. They also provide a foundation for discovery and optimization of drugs that poison bacterial Top1 using standard high-throughput approaches.
Assuntos
Proteínas de Bactérias/análise , Fracionamento Celular/métodos , Adutos de DNA/análise , DNA Topoisomerases Tipo I/análise , Ensaio de Imunoadsorção Enzimática/métodos , Ensaios de Triagem em Larga Escala/métodos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/isolamento & purificação , Adutos de DNA/isolamento & purificação , DNA Topoisomerases Tipo I/isolamento & purificação , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Immunoblotting/métodos , Mycobacterium smegmatis/química , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Reprodutibilidade dos Testes , Yersinia pestis/genéticaRESUMO
This work describes the synthesis of hybrid tetrahydro-1,5-naphthyridine and 1,5-naphthyridine derivatives fused with heterocycles such as chromenes and chromen-2-ones or coumarins, which were synthesized in good to high general yields. The synthetic route involves an intramolecular [4 + 2]-cycloaddition reaction of functionalized aldimines obtained by the condensation of 3-aminopyridine and aldehydes containing a double or triple carbon-carbon bond in orto position and allows the selective generation of three stereogenic centers in a short, efficient and reliable synthesis. The subsequent dehydrogenation of the fused tetrahydrochromeno[4,3-b][1,5]naphthyridines and/or tetrahydrochromeno[4,3-b][1,5]naphthyridin-6-ones leads to the formation of the corresponding tetracyclic chromeno[4,3-b][1,5]naphthyridine derivatives and/or chromeno[4,3-b][1,5]naphthyridin-6-ones in quantitative yields. Some of the prepared products showed activity as inhibitors of Topoisomerase I (TopI). Additionally, the cytotoxic behavior of these compounds has been studied in cell lines derived from human lung adenocarcinoma (A549) and human ovarian carcinoma (SKOV03), and on non-cancerous lung fibroblasts cell line (MRC5) where, on the last ones, the absence of cytotoxicity was observed. 7-Phenyl-6H-6a,7,12,12a-tetrahydrochromeno[4,3-b][1,5]naphthyridine 5a showed excellent cytotoxic activity with a IC50 value of 1.03⯱â¯0.30⯵M against the A549â¯cell line and a IC50 value of 1.75⯱â¯0.20⯵M against the SKOV03â¯cell line. The obtained results point to these compounds as good antiproliferative candidates.
Assuntos
Antineoplásicos/farmacologia , DNA Topoisomerases Tipo I/metabolismo , Naftiridinas/farmacologia , Inibidores da Topoisomerase I/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , DNA Topoisomerases Tipo I/isolamento & purificação , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Modelos Moleculares , Estrutura Molecular , Naftiridinas/síntese química , Naftiridinas/química , Saccharomyces cerevisiae/enzimologia , Relação Estrutura-Atividade , Inibidores da Topoisomerase I/síntese química , Inibidores da Topoisomerase I/química , Células Tumorais CultivadasRESUMO
Type IB DNA topoisomerases are enzymes to change the topological state of DNA molecules and are essential in studying replication, transcription, and recombination of nucleic acids in vitro. DNA topoisomerase IB from Vaccinia virus (vTopIB) is a 32 kDa, type I eukaryotic topoisomerase, which relaxed positively and negatively supercoiled DNAs without Mg2+ and ATP. Although vTopIB has been effectively produced in E. coli expression system, no studies remain available to explore an alternative platform to express recombinant vTopIB (rvTopIB) in a higher eukaryote, where the one can expect post-translational modifications that affect the activity of rvTopIB. Here in this study, rvTopIB with N-terminal tags was constructed and expressed in a silkworm-baculovirus expression vector system (silkworm-BEVS). We developed a simple two consecutive chromatography purification to obtain highly pure rvTopIB. The final yield of rvTopIB obtained from a baculovirus-infected silkworm larva was 83.25 µg. We also evaluated the activity and function of rvTopIB by the DNA relaxation activity assays using a negatively supercoiled pUC19 plasmid DNA as a substrate. With carefully assessing optimized conditions for the reaction buffer, we found that divalent ions, Mg2+, Mn2+, Ca2+, as well as ATP stimulate the DNA relaxation activity by rvTopIB. The functional and active form of rvTopIB, together with the yields of the protein we obtained, suggests that silkworm-BEVS would be a potential alternative platform to produce eukaryotic topoisomerases on an industrial scale.
Assuntos
DNA Topoisomerases Tipo I/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Vaccinia virus , Proteínas Virais/isolamento & purificação , Animais , Baculoviridae/genética , Bombyx/metabolismo , Bombyx/virologia , DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , Magnésio/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vaccinia virus/enzimologia , Vaccinia virus/genética , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
In the current study, we describe a novel DNA sensor system for specific and quantitative detection of mycobacteria, which is the causative agent of tuberculosis. Detection is achieved by using the enzymatic activity of the mycobacterial encoded enzyme topoisomerase IA (TOP1A) as a biomarker. The presented work is the first to describe how the catalytic activities of a member of the type IA family of topoisomerases can be exploited for specific detection of bacteria. The principle for detection relies on a solid support anchored DNA substrate with dual functions namely: (1) the ability to isolate mycobacterial TOP1A from crude samples and (2) the ability to be converted into a closed DNA circle upon reaction with the isolated enzyme. The DNA circle can act as a template for rolling circle amplification generating a tandem repeat product that can be visualized at the single molecule level by fluorescent labelling. This reaction scheme ensures specific, sensitive, and quantitative detection of the mycobacteria TOP1A biomarker as demonstrated by the use of purified mycobacterial TOP1A and extracts from an array of non-mycobacteria and mycobacteria species. When combined with mycobacteriophage induced lysis as a novel way of effective yet gentle extraction of the cellular content from the model Mycobacterium smegmatis, the DNA sensor system allowed detection of mycobacteria in small volumes of cell suspensions. Moreover, it was possible to detect M. smegmatis added to human saliva. Depending on the composition of the sample, we were able to detect 0.6 or 0.9 million colony forming units (CFU) per mL of mycobacteria, which is within the range of clinically relevant infection numbers. We, therefore, believe that the presented assay, which relies on techniques that can be adapted to limited resource settings, may be the first step towards the development of a new point-of-care diagnostic test for tuberculosis.
Assuntos
Proteínas de Bactérias/análise , Técnicas Biossensoriais/métodos , DNA Topoisomerases Tipo I/análise , Ácidos Nucleicos Imobilizados/metabolismo , Mycobacterium/isolamento & purificação , Patologia Molecular/métodos , Proteínas de Bactérias/metabolismo , Biomarcadores/análise , Biomarcadores/metabolismo , DNA Topoisomerases Tipo I/isolamento & purificação , DNA Topoisomerases Tipo I/metabolismo , Corantes Fluorescentes/química , Humanos , Ácidos Nucleicos Imobilizados/química , Mycobacterium/enzimologia , Sensibilidade e EspecificidadeRESUMO
We describe a gentle and rapid method to purify the intact multiprotein DNA replication complex using free flow electrophoresis (FFE). In particular, we applied FFE to purify the human cell DNA synthesome, which is a multiprotein complex that is fully competent to carry-out all phases of the DNA replication process in vitro using a plasmid containing the simian virus 40 (SV40) origin of DNA replication and the viral large tumor antigen (T-antigen) protein. The isolated native DNA synthesome can be of use in studying the mechanism by which mammalian DNA replication is carried-out and how anti-cancer drugs disrupt the DNA replication or repair process. Partially purified extracts from HeLa cells were fractionated in a native, liquid based separation by FFE. Dot blot analysis showed co-elution of many proteins identified as part of the DNA synthesome, including proliferating cell nuclear antigen (PCNA), DNA topoisomerase I (topo I), DNA polymerase δ (Pol δ), DNA polymerase É (Pol É), replication protein A (RPA) and replication factor C (RFC). Previously identified DNA synthesome proteins co-eluted with T-antigen dependent and SV40 origin-specific DNA polymerase activity at the same FFE fractions. Native gels show a multiprotein PCNA containing complex migrating with an apparent relative mobility in the megadalton range. When PCNA containing bands were excised from the native gel, mass spectrometric sequencing analysis identified 23 known DNA synthesome associated proteins or protein subunits.
Assuntos
DNA Polimerase Dirigida por DNA/análise , DNA Polimerase Dirigida por DNA/isolamento & purificação , Eletroforese/métodos , Complexos Multienzimáticos/análise , Complexos Multienzimáticos/isolamento & purificação , Antígenos Virais de Tumores/genética , Extratos Celulares/química , Linhagem Celular Tumoral , DNA Polimerase I/isolamento & purificação , DNA Polimerase II/isolamento & purificação , DNA Polimerase III/isolamento & purificação , Replicação do DNA , DNA Topoisomerases Tipo I/isolamento & purificação , Células HeLa , Humanos , Antígeno Nuclear de Célula em Proliferação/análise , Origem de Replicação/genética , Proteína de Replicação A/isolamento & purificação , Proteína de Replicação C/isolamento & purificação , Vírus 40 dos Símios/genéticaRESUMO
Ribonucleotides are the most abundant non-canonical component of yeast genomic DNA and their persistence is associated with a distinctive mutation signature characterized by deletion of a single repeat unit from a short tandem repeat. These deletion events are dependent on DNA topoisomerase I (Top1) and are initiated by Top1 incision at the relevant ribonucleotide 3'-phosphodiester. A requirement for the re-ligation activity of Top1 led us to propose a sequential cleavage model for Top1-dependent mutagenesis at ribonucleotides. Here, we test key features of this model via parallel in vitro and in vivo analyses. We find that the distance between two Top1 cleavage sites determines the deletion size and that this distance is inversely related to the deletion frequency. Following the creation of a gap by two Top1 cleavage events, the tandem repeat provides complementarity that promotes realignment to a nick and subsequent Top1-mediated ligation. Complementarity downstream of the gap promotes deletion formation more effectively than does complementarity upstream of the gap, consistent with constraints to realignment of the strand to which Top1 is covalently bound. Our data fortify sequential Top1 cleavage as the mechanism for ribonucleotide-dependent deletions and provide new insight into the component steps of this process.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , Ribonucleotídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Deleção de Sequência , Sequência de Bases , DNA/metabolismo , DNA Topoisomerases Tipo I/isolamento & purificação , Mutação da Fase de Leitura/genética , Sequências Repetitivas de Ácido Nucleico/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificaçãoRESUMO
Type IA topoisomerases represent promising antibacterial drug targets. Data exists suggesting that the two bacterial type IA topoisomerase enzymes-topoisomerase I and topoisomerase III-share an overlapping biological role. Furthermore, topoisomerase I has been shown to be essential for the survival of certain organisms lacking topoisomerase III. With this in mind, it is plausible that topoisomerase I may represent a potential target for selective antibacterial drug development. As many reported bacterial topoisomerase I purification protocols have either suffered from relatively low yield, numerous steps, or a simple failure to report target protein yield altogether, a high-yield and high-purity bacterial topoisomerase I expression and purification protocol is highly desirable. The goal of this study was therefore to optimize the expression and purification of topoisomerase I from Streptococcus mutans, a clinically relevant organism that plays a significant role in oral and extra-oral infection, in order to quickly and easily attain the requisite quantities of pure target enzyme suitable for use in assay development, compound library screening, and carrying out further structural and biochemical characterization analyses. Herein we report the systematic implementation and analysis of various expression and purification techniques leading to the development and optimization of a rapid and straightforward protocol for the auto-induced expression and two-step, affinity tag purification of Streptococcus mutans topoisomerase I yielding >20 mg/L of enzyme at over 95% purity.
Assuntos
Proteínas de Bactérias , DNA Topoisomerases Tipo I , Expressão Gênica , Streptococcus mutans/enzimologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , DNA Topoisomerases Tipo I/biossíntese , DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/isolamento & purificaçãoRESUMO
The different steps of the topoisomerase I catalytic cycle have been analyzed in the presence of the plant alkaloid thaspine (1- (2-(Dimethylamino)ethyl)-3,8-dimethoxychromeno[5,4,3-cde]chromene-5,10-dione), known to induce apoptosis in colon carcinoma cells. The experiments indicate that thaspine inhibits both the cleavage and the religation steps of the enzyme reaction. The inhibition is reversible and the effect is enhanced upon pre-incubation. Molecular docking simulations of thaspine over topoisomerase I, in the presence or absence of the DNA substrate, show that thaspine, when interacting with the enzyme alone in the closed or in the open state, can bind in proximity of the active residues preventing the cleavage reaction, whilst when docked with the enzyme-DNA cleavable complex intercalates between the DNA bases in a way similar to that found for camptothecin, explaining its religation inhibition. These results unequivocally demonstrate that thaspine targets human topoisomerase I .
Assuntos
Alcaloides/farmacologia , Antineoplásicos/farmacologia , Produtos Biológicos/farmacologia , DNA Topoisomerases Tipo I/metabolismo , Inibidores da Topoisomerase I/farmacologia , Alcaloides/química , Antineoplásicos/química , Produtos Biológicos/química , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/isolamento & purificação , Humanos , Cinética , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Inibidores da Topoisomerase I/químicaRESUMO
DNA topoisomerases play critical roles in regulating DNA topology and are essential enzymes for cell survival. In this study, a gene encoding type IA DNA topoisomerase was cloned from Staphylococcus aureus (S. aureus) sp. strain C-66, and the biochemical properties of recombinant enzyme was characterized. The nucleotide sequence analysis showed that the cloned gene contained an open reading frame (2070 bp) that could encode a polypeptide of 689 amino acids. The cloned gene actually produced 79.1 kDa functional enzyme (named Sau-TopoI) in Escherichia coli (E. coli). Sau-TopoI enzyme purified from E. coli showed ATP-independent and Mg(2+)-dependent manners for relaxing negatively supercoiled DNA. The relaxation activity of Sau-TopoI was inhibited by camptothecin, but not by nalidixic acid and etoposide. Cleavage site mapping showed that the enzyme could preferentially bind to and cleave the sequence GGNN↓CAT (N and ↓ represent any nucleotide and cleavage site, respectively). All these results suggest that the purified enzyme is type IA DNA topoisomerase. In addition, domain mapping analysis showed that the enzyme was composed of conserved four domains (I through IV), together with a variable C-terminal region containing a unique domain V.
Assuntos
DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/genética , Staphylococcus aureus/enzimologia , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Cátions Bivalentes/metabolismo , Clonagem Molecular , DNA Topoisomerases Tipo I/isolamento & purificação , DNA Topoisomerases Tipo I/metabolismo , Escherichia coli/citologia , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Temperatura , Fatores de Tempo , Inibidores da Topoisomerase I/farmacologiaRESUMO
On the basis of the asymmetrical charge distribution of Escherichia coli DNA topoisomerase I, we developed a new procedure to purify E. coli DNA topoisomerase I in the milligram range. The new procedure includes using both cation- and anion-exchange columns, i.e., SP-Sepharose FF and Q-Sepharose FF columns. The E. coli DNA topoisomerase I purified here is free of DNase contamination. The kinetic constants of the DNA relaxation reaction of E. coli DNA topoisomerase I were also determined.
Assuntos
Proteínas de Bactérias/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Escherichia coli/enzimologia , Proteínas Recombinantes/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Cromatografia por Troca Iônica , Clonagem Molecular , DNA/metabolismo , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/isolamento & purificação , Desoxirribonucleases/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Expressão Gênica , Cinética , Plasmídeos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Escherichia coli topoisomerase I (TopA) cleaves and rejoins one strand of double-stranded DNA to relax the negatively supercoiled DNA. Structurally, TopA contains an N-terminal catalytic fragment and a C-terminal zinc-binding region that is required for relaxation of the negatively supercoiled DNA. Here we report that E. coli TopA is an iron and zinc binding protein. The UV-Vis absorption measurements and metal content analyses reveal that TopA purified from E. coli cells grown in the rich LB medium contains both iron and zinc. However, TopA purified from E. coli cells grown in the M9 minimal medium has negligible amounts of zinc or iron and no topoisomerase activity. Nevertheless, supplement of exogenous zinc or iron in E. coli cells grown in the M9 minimal medium produces the zinc- or iron-bound TopA, respectively. Whereas the zinc-bound TopA is fully active to relax the negatively supercoiled DNA, the iron-bound TopA has little or no enzyme activity. Furthermore, excess iron in the M9 minimal medium is able to compete with the zinc binding in TopA in E. coli cells and attenuate the topoisomerase activity, suggesting that E. coli TopA may be modulated by iron and zinc binding in vivo.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , Escherichia coli/enzimologia , Ferro/metabolismo , Zinco/metabolismo , Sítios de Ligação , DNA Topoisomerases Tipo I/isolamento & purificação , Escherichia coli/citologia , Espectrofotometria UltravioletaRESUMO
BACKGROUND: Mycobacterium tuberculosis DNA topoisomerase I is an attractive target for discovery of novel TB drugs that act by enhancing the accumulation of the topoisomerase-DNA cleavage product. It shares a common transesterification domain with other type IA DNA topoisomerases. There is, however, no homology between the C-terminal DNA binding domains of Escherichia coli and M. tuberculosis DNA topoisomerase I proteins. RESULTS: A new protocol for expression and purification of recombinant M. tuberculosis DNA topoisomerase I (MtTOP) has been developed to produce enzyme of much higher specific activity than previously characterized recombinant enzyme. MtTOP was found to be less efficient than E. coli DNA topoisomerase I (EcTOP) in removal of remaining negative supercoils from partially relaxed DNA. DNA cleavage by MtTOP was characterized for the first time. Comparison of DNA cleavage site selectivity with EcTOP showed differences in cleavage site preferences, but the preferred sites of both enzymes have a C nucleotide in the -4 position. CONCLUSION: Recombinant M. tuberculosis DNA topoisomerase I can be expressed as a soluble protein and purified in high yield from E. coli host with a new protocol. Analysis of DNA cleavage with M. tuberculosis DNA substrate showed that the preferred DNA cleavage sites have a C nucleotide in the -4 position.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , DNA Topoisomerases Tipo I/isolamento & purificação , DNA Topoisomerases Tipo I/metabolismo , Expressão Gênica , Mycobacterium tuberculosis/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Escherichia coli/química , Escherichia coli/enzimologia , Escherichia coli/genética , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Histone chaperones are a group of histone-interacting proteins, involved in several important cellular functions. These chaperones are essential to facilitate ordered assembly of nucleosomes, both in replication dependent and independent manner. Replication independent function of histone chaperone is necessary for histone eviction during transcriptional initiation and elongation. In this chapter we have discussed a method to evaluate the role of histone chaperone NPM1 (the only known chaperone to get acetylated with functional consequence) in the transcriptional activation which is acetylation dependent.
Assuntos
Cromatina/genética , Histonas/metabolismo , Biologia Molecular/métodos , Chaperonas Moleculares/metabolismo , Transcrição Gênica , Acetilação , Animais , Bioensaio , Extratos Celulares , Núcleo Celular/metabolismo , Montagem e Desmontagem da Cromatina , DNA Topoisomerases Tipo I/isolamento & purificação , Drosophila melanogaster , Células HeLa , Histonas/isolamento & purificação , Humanos , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Camundongos , Nuclease do Micrococo/metabolismo , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Nucleofosmina , Plasmídeos/isolamento & purificação , Ligação Proteica , Moldes GenéticosRESUMO
The use of co-immunoprecipitation (co-IP) to purify multi-protein complexes has contributed greatly to our understanding of the DNA damage response network associated with Fanconi anemia (FA), Bloom syndrome (BS) and breast cancer. Four new FA genes and two new protein partners for the Bloom syndrome gene product have been identified by co-IP. Here, we discuss our experience in using co-IP and other techniques to isolate and characterize new FA and BS-related proteins.
Assuntos
Síndrome de Bloom/genética , Proteínas de Transporte/análise , Dano ao DNA , DNA Topoisomerases Tipo I/análise , Proteínas de Ligação a DNA/análise , Anemia de Fanconi/genética , Proteínas Nucleares/análise , RecQ Helicases/análise , Síndrome de Bloom/metabolismo , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , DNA Topoisomerases Tipo I/isolamento & purificação , DNA Topoisomerases Tipo I/metabolismo , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Anemia de Fanconi/metabolismo , Humanos , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , RecQ Helicases/isolamento & purificação , RecQ Helicases/metabolismoRESUMO
In this report we show that human DNA Topoisomerase I (Top1) forms DNA-protein adducts with nicked and gapped DNA structures lacking a conventional Top1 cleavage site. The radioactively labeled crosslinking products were identified by SDS-gel electrophoresis. The chemical structure of the groups at 5' or 3' end of the nick does not have an effect on the formation of these covalent adducts. Therefore, all kinds of nicks, either directly induced by ionizing radiation or reactive oxygen species or indirectly induced in the course of base excision repair (BER) are targets for Top1 that competes with BER proteins and other nick-sensors. Top1-DNA covalent adducts formed in cells exposed to DNA damaging agents can promote genetic instability.
Assuntos
Adutos de DNA/isolamento & purificação , Reparo do DNA , DNA Topoisomerases Tipo I/isolamento & purificação , DNA/química , Reagentes de Ligações Cruzadas/química , Quebras de DNA de Cadeia Simples , HumanosRESUMO
Kinetoplastid topoisomerase IB is an unusual bisubunit enzyme where reconstitution of the large (LdTOPIL or L) and small (LdTOPIS or S) subunits shows functional activity. It is yet to be deciphered whether one subunit or both navigate the heterodimer to its cellular DNA targets. Tethering a specific DNA-binding protein to topoisomerase I alters its site specificity. The chimeric constructs UMSBP-LdTOPIL/S or U-L/S (fusion of UMSBP to the N-terminus of L and reconstituted with S) and LdTOPIL/UMSBP-LdTOPIS or L/U-S (fusion of UMSBP to the N-terminus of S and reconstituted with L) exhibit relaxation activity. Only U-L/S shows altered site specificity and enhanced DNA-binding affinity for the universal minicircle sequence (UMS) containing substrate. This proves that L alone serves as the 'molecular steer' for this heterodimer. Reconstituted U-L/S also induces cleavage close to UMS and causes minicircle linearization. The differential properties of the reconstituted chimeras U-L/S and L/U-S reveal the structural and functional asymmetry between the heterodimer. Therefore this study helps in a better understanding of the mechanistic details underlying topoisomerization by this bi-subunit enzyme.
Assuntos
DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/metabolismo , Leishmania donovani/enzimologia , Proteínas de Protozoários/metabolismo , Animais , Anisotropia , Sequência de Bases , Camptotecina/farmacologia , Clonagem Molecular , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/isolamento & purificação , DNA Super-Helicoidal/química , DNA Super-Helicoidal/efeitos dos fármacos , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Estabilidade Enzimática/efeitos dos fármacos , Cloreto de Potássio/metabolismo , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/isolamento & purificação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por SubstratoRESUMO
Leishmania donovani, the causative organism of visceral leishmaniasis, contains a unique heterodimeric DNA topoisomerase IB (LdTop1). The catalytically active enzyme consists of a large subunit (LdTop1L), which contains the non-conserved N-terminal end and a phylogenetically conserved core domain, and of a small subunit (LdTop1S) which harbours the C-terminal region with a characteristic tyrosine residue in the active site. Heterologous co-expression of LdTop1L and LdTop1S in a topoisomerase I deficient yeast strain, reconstitutes a fully functional enzyme which can be used for structural studies. The role played by the non-conserved N-terminal extension of LdTop1S in both relaxation activity and CPT sensitivity of LdTop1 has been examined co-expressing the full-length LdTop1L with several deletions of LdTop1S lacking growing sequences of the N-terminal end. The sequential deletion study shows that the first 174 amino acids of LdTop1S are dispensable in terms of relaxation activity and DNA cleavage. It is also described that the trapping of the covalent complex between LdTop1 and DNA by CPT requires a pentapeptide between amino acid residues 175 and 179 of LdTop1S. Our results suggest the crucial role played by the N-terminal extension of the small subunit of DNA topoisomerase I.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , DNA/metabolismo , Leishmania donovani/enzimologia , Subunidades Proteicas/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Camptotecina/farmacologia , Clonagem Molecular , DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/isolamento & purificação , Relação Dose-Resposta a Droga , Deleção de Genes , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/isolamento & purificação , Saccharomyces cerevisiae/metabolismoRESUMO
BACKGROUND: The condensed state of the human sperm's chromatin is essential for the compact structure of the spermatozoon head, which is important for the fertilization process. The enzymes DNA topoisomerases (topo I and topo II) are responsible for the topological structure of the chromatin in somatic cells. The activities and the characterization of topoisomerases in mature human sperm cells have not been previously investigated. METHODS: Sperm cells were purified from the semen of healthy donors by standard procedures and assays measuring the activities, protein size and sensitivity to inhibitors of topoisomerases were performed. RESULTS: Topo I and topo II DNA relaxation activities are present in nuclear extracts derived from human sperm. The sperm topo I activity is inhibited by camptothecin, similarly to the somatic enzyme. An 85 kDa sperm protein, compared with the 100 kDa somatic topo IB enzyme, reacted with anti-human topo I antibody. Sperm topo II lacks the DNA decatenation activity of the somatic enzyme and a 97 kDa protein, compared with the 170 kDa somatic topo IIalpha enzyme, was detected with anti-human topo II antibody. Sperm nuclear extracts contained inhibitors of somatic topo II decatenation activity. CONCLUSIONS: Topoisomerase I and II activities as well as topo I and topo II proteins are present in mature human sperm cells. These enzymes possess unique properties compared with their somatic counterparts.
Assuntos
DNA Topoisomerases Tipo II/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Espermatozoides/enzimologia , Camptotecina/farmacologia , DNA Topoisomerases Tipo I/imunologia , DNA Topoisomerases Tipo I/isolamento & purificação , DNA Topoisomerases Tipo II/imunologia , DNA Topoisomerases Tipo II/isolamento & purificação , Etoposídeo/farmacologia , Humanos , Masculino , Peso Molecular , Inibidores da Topoisomerase I , Inibidores da Topoisomerase IIRESUMO
Reverse gyrases are topoisomerases that catalyze ATP-dependent positive supercoiling of circular covalently closed DNA. They consist of an N-terminal helicase-like domain, fused to a C-terminal topoisomerase I-like domain. Most of our knowledge on reverse gyrase-mediated positive DNA supercoiling is based on studies of archaeal enzymes. To identify general and individual properties of reverse gyrases, we set out to characterize the reverse gyrase from a hyperthermophilic eubacterium. Thermotoga maritima reverse gyrase relaxes negatively supercoiled DNA in the presence of ADP or the non-hydrolyzable ATP-analog ADPNP. Nucleotide binding is necessary, but not sufficient for the relaxation reaction. In the presence of ATP, positive supercoils are introduced at temperatures above 50 degrees C. However, ATP hydrolysis is stimulated by DNA already at 37 degrees C, suggesting that reverse gyrase is not frozen at this temperature, but capable of undergoing inter-domain communication. Positive supercoiling by reverse gyrase is strictly coupled to ATP hydrolysis. At the physiological temperature of 75 degrees C, reverse gyrase binds and hydrolyzes ATPgammaS. Surprisingly, ATPgammaS hydrolysis is stimulated by DNA, and efficiently promotes positive DNA supercoiling, demonstrating that inter-domain communication during positive supercoiling is fully functional with both ATP and ATPgammaS. These findings support a model for communication between helicase-like and topoisomerase domains in reverse gyrase, in which an ATP and DNA-induced closure of the cleft in the helicase-like domain initiates a cycle of conformational changes that leads to positive DNA supercoiling.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Proteínas de Bactérias/metabolismo , DNA Topoisomerases Tipo I/metabolismo , DNA Super-Helicoidal/metabolismo , Conformação de Ácido Nucleico , Thermotoga maritima , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/isolamento & purificação , DNA Super-Helicoidal/química , Modelos Moleculares , Mutação , Nucleotídeos/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Thermotoga maritima/enzimologia , Thermotoga maritima/genéticaRESUMO
The prostate-specific homeodomain protein NKX3.1 is a tumor suppressor that is commonly down-regulated in human prostate cancer. Using an NKX3.1 affinity column, we isolated topoisomerase I (Topo I) from a PC-3 prostate cancer cell extract. Topo I is a class 1B DNA-resolving enzyme that is ubiquitously expressed in higher organisms and many prokaryotes. NKX3.1 interacts with Topo I to enhance formation of the Topo I-DNA complex and to increase Topo I cleavage of DNA. The two proteins interacted in affinity pull-down experiments in the presence of either DNase or RNase. The NKX3.1 homeodomain was essential, but not sufficient, for the interaction with Topo I. NKX3.1 binding to Topo I occurred independently of the Topo I NH2-terminal domain. The binding of equimolar amounts of Topo I to NKX3.1 caused displacement of NKX3.1 from its cognate DNA recognition sequence. Topo I activity in prostates of Nkx3.1+/- and Nkx3.1-/- mice was reduced compared with wild-type mice, whereas Topo I activity in livers, where no NKX3.1 is expressed, was independent of Nkx3.1 genotype. Endogenous Topo I and NKX3.1 could be coimmunoprecipitated from LNCaP cells, where NKX3.1 and Topo I were found to colocalize in the nucleus and comigrate within the nucleus in response to either gamma-irradiation or mitomycin C exposure, two DNA-damaging agents. This is the first report that a homeodomain protein can modify the activity of Topo I and may have implications for organ-specific DNA replication, transcription, or DNA repair.