Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 23
Filtrar
1.
ACS Appl Mater Interfaces ; 13(8): 9482-9490, 2021 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-33476120

RESUMO

Solid-state nanopores show special potential as a new single-molecular characterization for nucleic acid assemblies and molecular machines. However, direct recognition of small dimensional species is still quite difficult due the lower resolution compared with biological pores. We recently reported a very efficient noise-reduction and resolution-enhancement mechanism via introducing high-dielectric additives (e.g., formamide) into conical glass nanopore (CGN) test buffer. Based on this advance, here, for the first time, we apply a bare CGN to directly recognize small dimensional assemblies induced by small molecules. Cocaine and its split aptamer (Capt assembly) are chosen as the model set. By introducing 20% formamide into CGN test buffer, high cocaine-specific distinguishing of the 113 nt Capt assembly has been realized without any covalent label or additional signaling strategies. The signal-to-background discrimination is much enhanced compared with control characterizations such as gel electrophoresis and fluorescence resonance energy transfer (FRET). As a further innovation, we verify that low-noise CGN can also enhance the resolution of small conformational/size changes happening on the side chain of large dimensional substrates. Long duplex concatamers generated from the hybridization chain reaction (HCR) are selected as the model substrates. In the presence of cocaine, low-noise CGN has sensitively captured the current changes when the 26 nt aptamer segment is assembled on the side chain of HCR duplexes. This paper proves that the introduction of the low-noise mechanism has significantly improved the resolution of the solid-state nanopore at smaller and finer scales and thus may direct extensive and deeper research in the field of CGN-based analysis at both single-molecular and statistical levels, such as molecular recognition, assembly characterization, structure identification, information storage, and target index.


Assuntos
Substâncias Macromoleculares/análise , Nanoporos , Aptâmeros de Nucleotídeos/análise , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Cocaína/metabolismo , DNA Concatenado/análise , DNA Concatenado/genética , DNA Concatenado/metabolismo , Eletroforese , Formamidas/química , Substâncias Macromoleculares/química , Hibridização de Ácido Nucleico/efeitos dos fármacos
2.
Mater Sci Eng C Mater Biol Appl ; 108: 110426, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31923928

RESUMO

A DNA fragment amplification/expression technology for the production of new generation biomaterials for scientific, industrial and biomedical applications is described. The technology enables the formation of artificial Open Reading Frames (ORFs) encoding concatemeric RNAs and proteins. It recruits the Type IIS SapI restriction endonuclease (REase) for an assembling of DNA fragments in an ordered head-to-tail-orientation. The technology employs a vector-enzymatic system, dedicated to the expression of newly formed, concatemeric ORFs from strong promoters. Four vector series were constructed to suit specialised needs. As a proof of concept, a model amplification of a 7-amino acid (aa) epitope from the S protein of HBV virus was performed, resulting in 500 copies of the epitope-coding DNA segment, consecutively linked and expressed in Escherichia coli (E. coli). Furthermore, a peptide with potential pro-regenerative properties (derived from an angiopoietin-related growth factor) was designed. Its aa sequence was back-translated, codon usage optimized and synthesized as a continuous ORF 10-mer. The 10-mer was cloned into the amplification vector, enabling the N-terminal fusion and multiplication of the encoded protein with MalE signal sequence. The obtained genes were expressed, and the proteins were purified. Conclusively, we show that the proteins are neither cytotoxic nor immunogenic and they have a very low allergic potential.


Assuntos
Materiais Biocompatíveis , DNA Concatenado , Escherichia coli , Expressão Gênica , Técnicas de Amplificação de Ácido Nucleico , Fases de Leitura Aberta , DNA Concatenado/genética , DNA Concatenado/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Vírus da Hepatite B/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Virais de Fusão/biossíntese , Proteínas Virais de Fusão/genética
3.
FEBS Open Bio ; 9(5): 840-850, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31034164

RESUMO

Connexins (Cx) are proteins that form cell-to-cell gap junction channels. A mutation at position 188 in the second extracellular loop (E2) domain of hCx46 has been linked to an autosomal dominant zonular pulverulent cataract. As it is dominantly inherited, it is possible that the mutant variant affects the co-expressed wild-type Cx and/or its interaction with other cellular components. Here, we proposed to use concatenated hCx46wt-hCx46N188T and hCx46N188T-hCx46wt to analyze how hCx46N188T affected co-expressed hCx46wt to achieve a dominant inheritance. Heterodimer hCx46wt-hCx46N188T formed fewer gap junction plaques compared to homodimer hCx46wt-hCx46wt, while the hCx46N188T-hCx46N188T homodimer formed almost no gap junction plaques. Dye uptake experiments showed that hemichannels of concatenated variants were similar to hemichannels of monomers. Molecular dynamics simulations revealed that for docking, the N188 of a protomer was engaged in hydrogen bonds (HBs) with R180, N189, and D191 of the counterpart protomer of the adjacent hemichannel. T188 suppressed the formation of HBs between protomers. Molecular dynamics simulations of an equimolar hCx46wt/hCx46N188T gap junction channel revealed a reduced number of HBs between protomers, suggesting reduction of gap junction channels between lens fibers co-expressing the variants.


Assuntos
Conexinas/genética , DNA Concatenado/genética , Simulação de Dinâmica Molecular , Mutação/genética , Catarata/congênito , Catarata/genética , Conexinas/metabolismo , DNA Concatenado/metabolismo , Junções Comunicantes/genética , Células HeLa , Humanos
4.
Nucleic Acids Res ; 47(5): 2169-2176, 2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30698805

RESUMO

Self-priming amplification of oligonucleotides is possible based on foldback of 3' ends, self-priming, and concatemerization, especially in the presence of phosphorothioate linkages. Such a simple replicative mechanism may have led to the accumulation of specific replicators at or near the origin of life. To determine how early replicators may have competed with one another, we have carried out selections with phosphorothiolated hairpins appended to a short random sequence library (N10). Upon the addition of deoxynucleoside triphosphates and a polymerase, concatemers quickly formed, and those random sequences that templated the insertion of purines, especially during initiation, quickly predominated. Over several serial transfers, particular sequences accumulated, and in isolation these were shown to outcompete less efficient replicators.


Assuntos
Primers do DNA/metabolismo , Replicação do DNA , Oligonucleotídeos/síntese química , Oligonucleotídeos/metabolismo , Sequência de Bases , Sequência Consenso , Primers do DNA/química , DNA Concatenado/síntese química , DNA Concatenado/química , DNA Concatenado/metabolismo , Evolução Molecular , Oligonucleotídeos/química , Origem da Vida , Moldes Genéticos
5.
Nucleic Acids Res ; 46(2): 861-872, 2018 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-29253195

RESUMO

DNA topoisomerases are essential enzymes involved in all the DNA processes and among them, type IA topoisomerases emerged as a key actor in the maintenance of genome stability. The hyperthermophilic archaeon, Sulfolobus solfataricus, contains three topoisomerases IA including one classical named TopA. SsoTopA is very efficient at unlinking DNA catenanes, grouping SsoTopA into the topoisomerase III family. SsoTopA is active over a wide range of temperatures and at temperatures of up to 85°C it produces highly unwound DNA. At higher temperatures, SsoTopA unlinks the two DNA strands. Thus depending on the temperature, SsoTopA is able to either prevent or favor DNA melting. While canonical topoisomerases III require a single-stranded DNA region or a nick in one of the circles to decatenate them, we show for the first time that a type I topoisomerase, SsoTopA, is able to efficiently unlink covalently closed catenanes, with no additional partners. By using single molecule experiments we demonstrate that SsoTopA requires the presence of a short single-stranded DNA region to be efficient. The unexpected decatenation property of SsoTopA probably comes from its high ability to capture this unwound region. This points out a possible role of TopA in S. solfataricus as a decatenase in Sulfolobus.


Assuntos
Proteínas Arqueais/metabolismo , DNA Topoisomerases Tipo I/metabolismo , DNA Catenado/metabolismo , Sulfolobus solfataricus/enzimologia , Proteínas Arqueais/genética , Sequência de Bases , DNA Topoisomerases Tipo I/genética , DNA Arqueal/química , DNA Arqueal/genética , DNA Arqueal/metabolismo , DNA Catenado/química , DNA Catenado/genética , DNA Concatenado/química , DNA Concatenado/genética , DNA Concatenado/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Temperatura Alta , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Sulfolobus solfataricus/genética
6.
J Virol ; 91(20)2017 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-28747509

RESUMO

Monomeric herpesvirus DNA is cleaved from concatemers and inserted into preformed capsids through the actions of the viral terminase. The terminase of herpes simplex virus (HSV) is composed of three subunits encoded by UL15, UL28, and UL33. The UL33-encoded protein (pUL33) interacts with pUL28, but its precise role in the DNA cleavage and packaging reaction is unclear. To investigate the function of pUL33, we generated a panel of recombinant viruses with either deletions or substitutions in the most conserved regions of UL33 using a bacterial artificial chromosome system. Deletion of 11 amino acids (residues 50 to 60 or residues 110 to 120) precluded viral replication, whereas the truncation of the last 10 amino acids from the pUL33 C terminus did not affect viral replication or the interaction of pUL33 with pUL28. Mutations that replaced the lysine at codon 110 and the arginine at codon 111 with alanine codons failed to replicate, and the pUL33 mutant interacted with pUL28 less efficiently. Interestingly, genomic termini of the large (L) and small (S) components were detected readily in cells infected with these mutants, indicating that concatemeric DNA was cleaved efficiently. However, the release of monomeric genomes as assessed by pulsed-field gel electrophoresis was greatly diminished, and DNA-containing capsids were not observed. These results suggest that pUL33 is necessary for one of the two viral DNA cleavage events required to release individual genomes from concatemeric viral DNA.IMPORTANCE This paper shows a role for pUL33 in one of the two DNA cleavage events required to release monomeric genomes from concatemeric viral DNA. This is the first time that such a phenotype has been observed and is the first identification of a function of this protein relevant to DNA packaging other than its interaction with other terminase components.


Assuntos
DNA Concatenado/metabolismo , DNA Viral/metabolismo , Genoma Viral , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiologia , Proteínas Virais/metabolismo , Montagem de Vírus , Animais , Linhagem Celular , Chlorocebus aethiops , Cromossomos Artificiais Bacterianos , Empacotamento do DNA , DNA Viral/genética , Eletroforese em Gel de Campo Pulsado , Herpesvirus Humano 1/enzimologia , Humanos , Células Vero , Proteínas Virais/genética , Replicação Viral
7.
Nucleic Acids Res ; 45(5): e29, 2017 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-27899664

RESUMO

Human embryonic stem cells (hESCs) are used as platforms for disease study, drug screening and cell-based therapy. To facilitate these applications, it is frequently necessary to genetically manipulate the hESC genome. Gene editing with engineered nucleases enables site-specific genetic modification of the human genome through homology-directed repair (HDR). However, the frequency of HDR remains low in hESCs. We combined efficient expression of engineered nucleases and integration-defective lentiviral vector (IDLV) transduction for donor template delivery to mediate HDR in hESC line WA09. This strategy led to highly efficient HDR with more than 80% of the selected WA09 clones harboring the transgene inserted at the targeted genomic locus. However, certain portions of the HDR clones contained the concatemeric IDLV genomic structure at the target site, probably resulted from recombination of the IDLV genomic input before HDR with the target. We found that the integrase protein of IDLV mediated the highly efficient HDR through the recruitment of a cellular protein, LEDGF/p75. This study demonstrates that IDLV-mediated HDR is a powerful and broadly applicable technology to carry out site-specific gene modification in hESCs.


Assuntos
Vetores Genéticos/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Integrases/genética , Lentivirus/genética , Reparo de DNA por Recombinação , Proteínas Virais/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Bases , Linhagem Celular , DNA Concatenado/genética , DNA Concatenado/metabolismo , Edição de Genes/métodos , Vetores Genéticos/química , Genoma Humano , Células-Tronco Embrionárias Humanas/citologia , Humanos , Integrases/metabolismo , Lentivirus/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Virais/metabolismo
8.
J Nanosci Nanotechnol ; 15(6): 4170-7, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26369026

RESUMO

Self-assembly of DNA concatemers from native duplexes and those containing non-nucleotidic bridges of varying polarity composed of repeating oligo(ethylene glycol) phosphates -O(CH2CH2O)(n)PO2- or α,Ω-alkanediol phosphates -O(CH2)10OPO2(-)- units was compared. The structures obtained were characterised by polyacrylamide gel electrophoresis, enzymatic digestion and AFM. Our results have revealed that chemically-modified duplexes favour self-termination of concatemer growth and yield up to 35% of nanosized DNA rings.


Assuntos
DNA Concatenado/química , Etilenoglicol/química , Nanoestruturas/química , Sequência de Bases , DNA Concatenado/metabolismo , Desoxirribonucleases/metabolismo , Microscopia de Força Atômica , Dados de Sequência Molecular , Oligonucleotídeos
9.
Mol Pharmacol ; 87(3): 401-9, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25519838

RESUMO

Activation of human ether-a-go-go-related gene 1 (hERG1) K(+) channels mediates repolarization of action potentials in cardiomyocytes. RPR-260243 [(3R,4R)-4-[3-(6-methoxy-quinolin-4-yl)-3-oxo-propyl]-1-[3-(2,3,5-trifluorophenyl)-prop-2-ynyl]-piperidine-3-carboxylic acid] (RPR) slows deactivation and attenuates inactivation of hERG1 channels. A detailed understanding of the molecular mechanism of hERG1 agonists such as RPR may facilitate the design of more selective and potent compounds for prevention of arrhythmia associated with abnormally prolonged ventricular repolarization. RPR binds to a hydrophobic pocket located between two adjacent hERG1 subunits, and, hence, a homotetrameric channel has four identical RPR binding sites. To investigate the stoichiometry of altered channel gating induced by RPR, we constructed and characterized tetrameric hERG1 concatemers containing a variable number of wild-type subunits and subunits containing a point mutation (L553A) that rendered the channel insensitive to RPR, ostensibly by preventing ligand binding. The slowing of deactivation by RPR was proportional to the number of wild-type subunits incorporated into a concatenated tetrameric channel, and four wild-type subunits were required to achieve maximal slowing of deactivation. In contrast, a single wild-type subunit within a concatenated tetramer was sufficient to achieve half of the maximal RPR-induced shift in the voltage dependence of hERG1 inactivation, and maximal effect was achieved in channels containing three or four wild-type subunits. Together our findings suggest that the allosteric modulation of channel gating involves distinct mechanisms of coupling between drug binding and altered deactivation and inactivation.


Assuntos
DNA Concatenado/química , DNA Concatenado/metabolismo , Canais de Potássio Éter-A-Go-Go/química , Canais de Potássio Éter-A-Go-Go/metabolismo , Ativação do Canal Iônico/fisiologia , Piperidinas/metabolismo , Quinolinas/metabolismo , Animais , Feminino , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Piperidinas/farmacologia , Ligação Proteica/fisiologia , Quinolinas/farmacologia , Estereoisomerismo , Xenopus laevis
10.
PLoS One ; 9(2): e89070, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24551219

RESUMO

HMGB1 is an architectural protein in chromatin, acting also as a signaling molecule outside the cell. Recent reports from several laboratories provided evidence that a number of both the intracellular and extracellular functions of HMGB1 may depend on redox-sensitive cysteine residues of the protein. In this study we demonstrate that redox state of HMGB1 can significantly modulate the ability of the protein to bind and bend DNA, as well as to promote DNA end-joining. We also report a high affinity binding of histone H1 to hemicatenated DNA loops and DNA minicircles. Finally, we show that reduced HMGB1 can readily displace histone H1 from DNA, while oxidized HMGB1 has limited capacity for H1 displacement. Our results suggested a novel mechanism for the HMGB1-mediated modulation of histone H1 binding to DNA. Possible biological consequences of linker histones H1 replacement by HMGB1 for the functioning of chromatin are discussed.


Assuntos
Cromatina/metabolismo , DNA Circular/metabolismo , DNA Concatenado/metabolismo , Proteína HMGB1/metabolismo , Histonas/metabolismo , Proteínas Recombinantes/metabolismo , Animais , Bovinos , Cromatina/genética , DNA Circular/genética , DNA Concatenado/genética , Expressão Gênica , Vetores Genéticos/química , Proteína HMGB1/genética , Histonas/genética , Humanos , Oxirredução , Ligação Proteica , Ratos , Proteínas Recombinantes/genética
12.
Nature ; 454(7202): 297-301, 2008 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-18596691

RESUMO

Sister chromatid cohesion, which is essential for mitosis, is mediated by a multi-subunit protein complex called cohesin. Cohesin's Scc1, Smc1 and Smc3 subunits form a tripartite ring structure, and it has been proposed that cohesin holds sister DNA molecules together by trapping them inside its ring. To test this, we used site-specific crosslinking to create chemical connections at the three interfaces between the three constituent polypeptides of the ring, thereby creating covalently closed cohesin rings. As predicted by the ring entrapment model, this procedure produced dimeric DNA-cohesin structures that are resistant to protein denaturation. We conclude that cohesin rings concatenate individual sister minichromosome DNA molecules.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Fúngicos/metabolismo , DNA Concatenado/metabolismo , DNA Fúngico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/química , Proteínas Cromossômicas não Histona/química , Estrutura Quaternária de Proteína/efeitos dos fármacos , Subunidades Proteicas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Dodecilsulfato de Sódio/farmacologia , Coesinas
13.
FEMS Microbiol Lett ; 284(1): 58-67, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18492061

RESUMO

The 46-kb plasmid pSK41 is the prototype of a family of staphylococcal conjugative multiresistance plasmids. Sequence analyses have revealed the presence of a putative resolvase gene, res, on pSK41, and identical or related genes carried by other staphylococcal multiresistance plasmids. Carriage of the res region was found to ameliorate the accumulation of multimeric plasmid forms, and recombinant plasmids encoding a wild-type res gene exhibited greater plasmid segregational stability than counterparts carrying a nonfunctional mutant, irrespective of whether the cognate or a heterologous replication system and host was utilized. In vitro DNA-binding studies demonstrated that purified Res protein binds within the intergenic region upstream of the res coding sequence. Six copies of an imperfect 11-bp repeat sequence were identified within DNA sequences protected by Res in DNAseI footprinting studies, in an arrangement that suggests a typical resolution site organization consisting of three subsites.


Assuntos
Replicação do DNA , Resolvases de Junção Holliday/genética , Fatores R , Staphylococcus aureus/genética , Proteínas de Bactérias/genética , Sequência de Bases , Sítios de Ligação , Pegada de DNA , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Concatenado/metabolismo , DNA Intergênico , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Resolvases de Junção Holliday/isolamento & purificação , Resolvases de Junção Holliday/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Recombinação Genética , Análise de Sequência de DNA , Staphylococcus aureus/fisiologia
14.
FEBS Lett ; 582(5): 666-72, 2008 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-18242185

RESUMO

This study provides evidence that proteasomal activity is required at multiple steps in human cytomegalovirus replication. Electron microscopy revealed that no viral particles were assembled in the presence of proteasome inhibitor MG132. Immunofluorescence and Western blot analyses using MG132 demonstrated that immediate early gene expression was suppressed at low but not high MOI. In contrast, expression of late proteins was completely blocked independent of MOI. Additionally, pulsed-field gel electrophoresis demonstrated that MG132 interferes with cleavage of HCMV DNA. Bromodeoxyuridine incorporation studies showed that de novo viral DNA synthesis is reduced in the presence of MG132. Furthermore, in contrast to previous hypotheses we demonstrated that neither the ND10 components PML and hDaxx nor NFkappaB activation represent the target for MG132.


Assuntos
Citomegalovirus/efeitos dos fármacos , Citomegalovirus/fisiologia , DNA Viral/biossíntese , Leupeptinas/farmacologia , Inibidores de Proteassoma , Montagem de Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Antivirais/farmacologia , Células Cultivadas , Infecções por Citomegalovirus/virologia , DNA Concatenado/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/virologia , Humanos , NF-kappa B/deficiência , NF-kappa B/metabolismo , Proteínas Virais/metabolismo , Vírion/efeitos dos fármacos , Vírion/metabolismo
15.
Nature ; 451(7176): 318-22, 2008 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-18202654

RESUMO

In nature, self-assembling and disassembling complexes of proteins and nucleic acids bound to a variety of ligands perform intricate and diverse dynamic functions. In contrast, attempts to rationally encode structure and function into synthetic amino acid and nucleic acid sequences have largely focused on engineering molecules that self-assemble into prescribed target structures, rather than on engineering transient system dynamics. To design systems that perform dynamic functions without human intervention, it is necessary to encode within the biopolymer sequences the reaction pathways by which self-assembly occurs. Nucleic acids show promise as a design medium for engineering dynamic functions, including catalytic hybridization, triggered self-assembly and molecular computation. Here, we program diverse molecular self-assembly and disassembly pathways using a 'reaction graph' abstraction to specify complementarity relationships between modular domains in a versatile DNA hairpin motif. Molecular programs are executed for a variety of dynamic functions: catalytic formation of branched junctions, autocatalytic duplex formation by a cross-catalytic circuit, nucleated dendritic growth of a binary molecular 'tree', and autonomous locomotion of a bipedal walker.


Assuntos
Simulação por Computador , DNA/química , DNA/metabolismo , Conformação de Ácido Nucleico , Biopolímeros/química , Biopolímeros/metabolismo , Catálise , DNA Concatenado/química , DNA Concatenado/metabolismo , Dendrímeros/química , Dendrímeros/metabolismo , Marcha , Cinética , Modelos Biológicos , Processos Estocásticos , Caminhada
16.
J Virol ; 81(13): 7034-40, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17459921

RESUMO

Cells infected by adenovirus E4 mutants accumulate end-to-end concatemers of the viral genome that are assembled from unit-length viral DNAs by nonhomologous end joining (NHEJ). Genome concatenation can be prevented by expression either of E4 11k (product of E4orf3) or of the complex of E4 34k (product of E4orf6) and E1b 55k. Both E4 11k and the E4 34k/E1b 55k complex prevent concatenation at least in part by inactivation of the host protein Mre11: E4 11k sequesters Mre11 in aggresomes, while the E4 34k/E1b 55k complex participates in a virus-specific E3 ubiquitin ligase that mediates ubiquitination and proteasomal degradation. The E4 34k/E1b 55k complex, but not E4 11k, also inhibits NHEJ activity on internal breaks in the viral genome and on V(D)J recombination substrate plasmids, suggesting that it may interfere with NHEJ independently of its effect on Mre11. We show here that DNA ligase IV, which performs the joining step of NHEJ, is degraded as a consequence of adenovirus infection. Degradation is dependent upon E4 34k and E1b 55k, functional proteasomes, and the activity of cellular cullin 5, a component of the adenoviral ubiquitin ligase. DNA ligase IV also interacts physically with E1b 55k. The data demonstrate that DNA ligase IV, like Mre11, is a substrate for the adenovirus-specific E3 ubiquitin ligase; identify an additional viral approach to prevention of genome concatenation; and provide a mechanism for the general inhibition of NHEJ by adenoviruses.


Assuntos
Infecções por Adenoviridae/metabolismo , Adenoviridae/metabolismo , Proteínas E1B de Adenovirus/metabolismo , DNA Ligases/metabolismo , DNA Concatenado/metabolismo , DNA Viral/metabolismo , Genoma Viral/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Adenoviridae/genética , Infecções por Adenoviridae/genética , Proteínas E1B de Adenovirus/genética , Proteínas E4 de Adenovirus , Linhagem Celular Tumoral , Quebras de DNA , DNA Ligase Dependente de ATP , DNA Ligases/genética , DNA Concatenado/genética , DNA Viral/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteína Homóloga a MRE11 , Complexo de Endopeptidases do Proteassoma/genética , Processamento de Proteína Pós-Traducional/fisiologia , Recombinação Genética/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
17.
Mitochondrion ; 7(1-2): 17-23, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17280877

RESUMO

Homoplasmy is a basic genetic state of mitochondria, in which all of the hundreds to thousands of mitochondrial (mt)DNA copies within a cell or an individual have the same nucleotide-sequence. It was recently found that "vegetative segregation" to generate homoplasmic cells is an active process under genetic control. In the yeast Saccharomyces cerevisiae, the Mhr1 protein which catalyzes a key reaction in mtDNA homologous recombination, plays a pivotal role in vegetative segregation. Conversely, within the nuclear genome, homologous DNA recombination causes genetic diversity. Considering these contradictory roles of this key reaction in DNA recombination, possible functions of homoplasmy are discussed.


Assuntos
DNA Mitocondrial/genética , Mitocôndrias/fisiologia , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Segregação de Cromossomos/fisiologia , Replicação do DNA , DNA Concatenado/metabolismo , DNA Mitocondrial/metabolismo , Mitocôndrias/genética , Recombinação Genética/fisiologia , Saccharomyces cerevisiae/metabolismo
18.
Mol Cell Biol ; 27(3): 1133-45, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17116696

RESUMO

Hypersuppressiveness, as observed in Saccharomyces cerevisiae, is an extremely biased inheritance of a small mitochondrial DNA (mtDNA) fragment that contains a replication origin (HS [rho(-)] mtDNA). Our previous studies showed that concatemers (linear head-to-tail multimers) are obligatory intermediates for mtDNA partitioning and are primarily formed by rolling-circle replication mediated by Mhr1, a protein required for homologous mtDNA recombination. In this study, we found that Mhr1 is required for the hypersuppressiveness of HS [ori5] [rho(-)] mtDNA harboring ori5, one of the replication origins of normal ([rho(+)]) mtDNA. In addition, we detected an Ntg1-stimulated double-strand break at the ori5 locus. Purified Ntg1, a base excision repair enzyme, introduced a double-stranded break by itself into HS [ori5] [rho(-)] mtDNA at ori5 isolated from yeast cells. Both hypersuppressiveness and concatemer formation of HS [ori5] [rho(-)] mtDNA are simultaneously suppressed by the ntg1 null mutation. These results support a model in which, like homologous recombination, rolling-circle HS [ori5] [rho(-)] mtDNA replication is initiated by double-stranded breakage in ori5, followed by Mhr1-mediated homologous pairing of the processed nascent DNA ends with circular mtDNA. The hypersuppressiveness of HS [ori5] [rho(-)] mtDNA depends on a replication advantage furnished by the higher density of ori5 sequences and on a segregation advantage furnished by the higher genome copy number on transmitted concatemers.


Assuntos
DNA Fúngico/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Herança Extracromossômica/genética , Recombinação Genética , Origem de Replicação/genética , Saccharomyces cerevisiae/genética , Aloxano/farmacologia , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Concatenado/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Modelos Genéticos , Dados de Sequência Molecular , N-Glicosil Hidrolases/metabolismo , Proteínas Nucleares/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Recombinação Genética/efeitos dos fármacos , Origem de Replicação/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Supressão Genética/efeitos dos fármacos , Fatores de Transcrição/metabolismo
19.
Mol Microbiol ; 52(2): 501-13, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15066036

RESUMO

The development of bacteriophage lambda and double-stranded DNA viruses in general involves the convergence of two separate pathways: DNA replication and head assembly. Clearly, packaging will proceed only if an empty capsid shell, the prohead, is present to receive the DNA, but genetic evidence suggests that proheads play another role in the packaging process. For example, lambda phages with an amber mutation in any head gene or in FI, the gene encoding the accessory packaging protein gpFI, are able to produce normal amounts of DNA concatemers but they are not cut, or matured, into unit length chromosomes for packaging. Similar observations have been made for herpes simplex 1 virus. In the case of lambda, a negative model proposes that in the amber phages, unassembled capsid components are inhibitory to maturation, and a positive model suggests that assembled proheads are required for cutting. We tested the negative model by using a deletion mutant devoid of all prohead genes and FI in an in vivo cos cleavage assay; in this deleted phage, the cohesive ends were not cut. When lambda proheads and gpFI were provided in vivo via a second prophage, cutting was restored, and gpFI was required, results that support the positive model. Phage 21 is a sister phage of lambda, and although its capsid proteins share approximately 60% residue identity with lambda's, phage 21 proheads did not restore cutting, even when provided with the accessory protein gpFI. Models for the role of proheads and gpFI in cos cutting are discussed.


Assuntos
Bacteriófago lambda/genética , Capsídeo/fisiologia , DNA Concatenado/metabolismo , DNA Viral/metabolismo , Endodesoxirribonucleases/metabolismo , Proteínas Virais/metabolismo , Sítios de Ligação , DNA Concatenado/genética , DNA Viral/genética , Modelos Genéticos , Mutação , Plasmídeos , Proteínas Virais/genética
20.
Mol Microbiol ; 51(4): 937-48, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14763971

RESUMO

Catalysis of DNA recombination by Tn3 resolvase is conditional on prior formation of a synapse, comprising 12 resolvase subunits and two recombination sites (res). Each res binds a resolvase dimer at site I, where strand exchange takes place, and additional dimers at two adjacent 'accessory' binding sites II and III. 'Hyperactive' resolvase mutants, that catalyse strand exchange at site I without accessory sites, were selected in E. coli. Some single mutants can resolve a res x site I plasmid (that is, with one res and one site I), but two or more activating mutations are necessary for efficient resolution of a site I x site I plasmid. Site I x site I resolution by hyperactive mutants can be further stimulated by mutations at the crystallographic 2-3' interface that abolish activity of wild-type resolvase. Activating mutations may allow regulatory mechanisms of the wild-type system to be bypassed, by stabilizing or destabilizing interfaces within and between subunits in the synapse. The positions and characteristics of the mutations support a mechanism for strand exchange by serine recombinases in which the DNA is on the outside of a recombinase tetramer, and the tertiary/quaternary structure of the tetramer is reconfigured.


Assuntos
Elementos de DNA Transponíveis , Transposon Resolvases/genética , Transposon Resolvases/metabolismo , Sítios de Ligação , Catálise , Domínio Catalítico , DNA/metabolismo , DNA Circular/metabolismo , DNA Concatenado/genética , DNA Concatenado/metabolismo , DNA Super-Helicoidal/metabolismo , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Rearranjo Gênico , Modelos Moleculares , Mutagênese , Mutação de Sentido Incorreto , Plasmídeos/genética , Plasmídeos/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Recombinação Genética , Transposon Resolvases/química
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA