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1.
Nucleic Acids Res ; 46(20): 10855-10869, 2018 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-30285153

RESUMO

Homologous recombination is essential to genome maintenance, and also to genome diversification. In virtually all organisms, homologous recombination depends on the RecA/Rad51-family recombinases, which catalyze ATP-dependent formation of homologous joints-critical intermediates in homologous recombination. RecA/Rad51 binds first to single-stranded (ss) DNA at a damaged site to form a spiral nucleoprotein filament, after which double-stranded (ds) DNA interacts with the filament to search for sequence homology and to form consecutive base pairs with ssDNA ('pairing'). How sequence homology is recognized and what exact role filament formation plays remain unknown. We addressed the question of whether filament formation is a prerequisite for homologous joint formation. To this end we constructed a nonpolymerizing (np) head-to-tail-fused RecA dimer (npRecA dimer) and an npRecA monomer. The npRecA dimer bound to ssDNA, but did not form continuous filaments upon binding to DNA; it formed beads-on-string structures exclusively. Although its efficiency was lower, the npRecA dimer catalyzed the formation of D-loops (a type of homologous joint), whereas the npRecA monomer was completely defective. Thus, filament formation contributes to efficiency, but is not essential to sequence-homology recognition and pairing, for which a head-to-tail dimer form of RecA protomer is required and sufficient.


Assuntos
DNA de Cadeia Simples/metabolismo , Recombinação Homóloga , Multimerização Proteica , Recombinases Rec A/fisiologia , Pareamento de Bases/fisiologia , Catálise , DNA de Cadeia Simples/química , Escherichia coli , Instabilidade Genômica/genética , Recombinação Homóloga/genética , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Multimerização Proteica/fisiologia , Recombinases Rec A/genética , Recombinases Rec A/metabolismo
2.
Nucleic Acids Res ; 45(1): 337-352, 2017 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-27794044

RESUMO

RecA-family recombinase-catalyzed ATP-dependent homologous joint formation is critical for homologous recombination, in which RecA or Rad51 binds first to single-stranded (ss)DNA and then interacts with double-stranded (ds)DNA. However, when RecA or Rad51 interacts with dsDNA before binding to ssDNA, the homologous joint-forming activity of RecA or Rad51 is quickly suppressed. We found that under these and adenosine diphosphate (ADP)-generating suppressive conditions for the recombinase activity, RecA or Rad51 at similar optimal concentrations enhances the DNA ligase-catalyzed dsDNA end-joining (DNA ligation) about 30- to 40-fold. The DNA ligation enhancement by RecA or Rad51 transforms most of the substrate DNA into multimers within 2-5 min, and for this enhancement, ADP is the common and best cofactor. Adenosine triphosphate (ATP) is effective for RecA, but not for Rad51. Rad51/RecA-enhanced DNA ligation depends on dsDNA-binding, as shown by a mutant, and is independent of physical interactions with the DNA ligase. These observations demonstrate the common and unique activities of RecA and Rad51 to juxtapose dsDNA-ends in preparation for covalent joining by a DNA ligase. This new in vitro function of Rad51 provides a simple explanation for our genetic observation that Rad51 plays a role in the fidelity of the end-joining of a reporter plasmid DNA, by yeast canonical non-homologous end-joining (NHEJ) in vivo.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , DNA Fúngico/genética , Rad51 Recombinase/genética , Recombinases Rec A/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Difosfato de Adenosina/metabolismo , Coenzimas/metabolismo , DNA/genética , DNA/metabolismo , DNA Fúngico/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Rad51 Recombinase/metabolismo , Recombinases Rec A/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
3.
J Stroke Cerebrovasc Dis ; 27(7): e132-e134, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29525082

RESUMO

Some stroke patients with the acute aortic dissection receiving thrombolysis treatment resulted in fatalities. Thus, the concurrent acute aortic dissection is the contraindication for the intravenous recombinant tissue-type plasminogen activator. However, the safety and the effectiveness of the intravenous recombinant tissue-type plasminogen activator therapy are not known in patients with stroke some days after acute aortic dissection treatment. Here, we first report a case of a man with a cardioembolism due to the nonvalvular atrial fibrillation, who received the intravenous recombinant tissue-type plasminogen activator therapy 117 days after the traumatic Stanford type A acute aortic dissection operation. Without the intravenous recombinant tissue-type plasminogen activator therapy, the prognosis was expected to be miserable. However, the outcome was good with no complication owing to the intravenous recombinant tissue-type plasminogen activator therapy. Our case suggests the effectiveness and the safety of the intravenous recombinant tissue-type plasminogen activator therapy to the ischemic stroke some days after acute aortic dissection treatment.


Assuntos
Dissecção Aórtica/cirurgia , Fibrinolíticos/uso terapêutico , Complicações Pós-Operatórias/tratamento farmacológico , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/etiologia , Ativador de Plasminogênio Tecidual/uso terapêutico , Administração Intravenosa , Idoso de 80 Anos ou mais , Dissecção Aórtica/complicações , Dissecção Aórtica/diagnóstico por imagem , Fibrilação Atrial/complicações , Fibrilação Atrial/diagnóstico por imagem , Fibrilação Atrial/cirurgia , Humanos , Masculino , Complicações Pós-Operatórias/diagnóstico por imagem , Proteínas Recombinantes/uso terapêutico , Acidente Vascular Cerebral/diagnóstico por imagem , Terapia Trombolítica
4.
J Stroke Cerebrovasc Dis ; 27(6): e110-e112, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29402614

RESUMO

Although foam sclerotherapy to varicose veins is now a popular treatment because of its high efficacy and safety, some neurologic complications have recently been reported. Presently, the effectiveness and safety of intravenous recombinant tissue-type plasminogen activator therapy to stroke following foam sclerotherapy remain unclear. Here, we report the case of a 68-year-old woman whose ischemic symptoms following foam sclerotherapy were treated by intravenous recombinant tissue-type plasminogen activator. After she was admitted, the venous thrombosis in her right soleus vein and a patent foramen ovale causing the right-to-left shunt were revealed. Thus, we diagnosed the ischemic symptoms were due to paradoxical embolism following foam sclerotherapy. After intravenous recombinant tissue-type plasminogen activator therapy, there was no complication and the outcome was good. Our case suggests the effectiveness and the safety of intravenous recombinant tissue-type plasminogen activator therapy to paradoxical embolism following foam sclerotherapy.


Assuntos
Embolia Paradoxal/tratamento farmacológico , Fibrinolíticos/uso terapêutico , Forame Oval Patente/complicações , Escleroterapia/efeitos adversos , Acidente Vascular Cerebral/tratamento farmacológico , Ativador de Plasminogênio Tecidual/uso terapêutico , Administração Intravenosa , Idoso , Embolia Paradoxal/etiologia , Feminino , Forame Oval Patente/terapia , Humanos , Polidocanol , Polietilenoglicóis/efeitos adversos , Polietilenoglicóis/uso terapêutico , Proteínas Recombinantes/uso terapêutico , Soluções Esclerosantes/efeitos adversos , Soluções Esclerosantes/uso terapêutico , Acidente Vascular Cerebral/etiologia , Varizes/complicações , Varizes/terapia , Trombose Venosa/complicações , Trombose Venosa/terapia
5.
Nucleic Acids Res ; 43(2): 973-86, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25561575

RESUMO

In all organisms, RecA-family recombinases catalyze homologous joint formation in homologous genetic recombination, which is essential for genome stability and diversification. In homologous joint formation, ATP-bound RecA/Rad51-recombinases first bind single-stranded DNA at its primary site and then interact with double-stranded DNA at another site. The underlying reason and the regulatory mechanism for this conserved binding order remain unknown. A comparison of the loop L1 structures in a DNA-free RecA crystal that we originally determined and in the reported DNA-bound active RecA crystals suggested that the aspartate at position 161 in loop L1 in DNA-free RecA prevented double-stranded, but not single-stranded, DNA-binding to the primary site. This was confirmed by the effects of the Ala-replacement of Asp-161 (D161A), analyzed directly by gel-mobility shift assays and indirectly by DNA-dependent ATPase activity and SOS repressor cleavage. When RecA/Rad51-recombinases interact with double-stranded DNA before single-stranded DNA, homologous joint-formation is suppressed, likely by forming a dead-end product. We found that the D161A-replacement reduced this suppression, probably by allowing double-stranded DNA to bind preferentially and reversibly to the primary site. Thus, Asp-161 in the flexible loop L1 of wild-type RecA determines the preference for single-stranded DNA-binding to the primary site and regulates the DNA-binding order in RecA-catalyzed recombinase reactions.


Assuntos
Reparo do DNA , Recombinação Homóloga , Recombinases Rec A/química , Trifosfato de Adenosina/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/metabolismo , Biocatálise , DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Ligação Proteica , Conformação Proteica , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Serina Endopeptidases/metabolismo
6.
J Exp Biol ; 219(Pt 8): 1146-53, 2016 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-26944491

RESUMO

To elucidate the degradation process of the posterior silk gland during metamorphosis of the silkworm ITALIC! Bombyx mori, tissues collected on the 6th day after entering the 5th instar (V6), prior to spinning (PS), during spinning (SP) and after cocoon formation (CO) were used to analyze macroautophagy, chaperone-mediated autophagy (CMA) and the adenosine triphosphate (ATP)-dependent ubiquitin proteasome. Immediately after entering metamorphosis stage PS, the levels of ATP and phosphorylated p70S6 kinase protein decreased spontaneously and continued to decline at SP, followed by a notable restoration at CO. In contrast, phosphorylated AMP-activated protein kinase α (AMPKα) showed increases at SP and CO. Most of the Atg8 protein was converted to form II at all stages. The levels of ubiquitinated proteins were high at SP and CO, and low at PS. The proteasome activity was high at V6 and PS but low at SP and CO. In the isolated lysosome fractions, levels of Hsc70/Hsp70 protein began to increase at PS and continued to rise at SP and CO. The lysosomal cathepsin B/L activity showed a dramatic increase at CO. Our results clearly demonstrate that macroautophagy occurs before entering the metamorphosis stage and strongly suggest that the CMA pathway may play an important role in the histolysis of the posterior silk gland during metamorphosis.


Assuntos
Estruturas Animais/metabolismo , Autofagia , Bombyx/anatomia & histologia , Metamorfose Biológica , Chaperonas Moleculares/metabolismo , Seda/metabolismo , Trifosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Estruturas Animais/anatomia & histologia , Estruturas Animais/efeitos dos fármacos , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagia/efeitos dos fármacos , Bombyx/efeitos dos fármacos , Bombyx/metabolismo , Glucose/análise , Hemolinfa/efeitos dos fármacos , Hemolinfa/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Larva/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Metamorfose Biológica/efeitos dos fármacos , Tamanho do Órgão/efeitos dos fármacos , Pressão Osmótica/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ubiquitina/metabolismo
7.
Nucleic Acids Res ; 42(2): 941-51, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24163251

RESUMO

The Saccharomyces cerevisiae Rad52 protein is essential for efficient homologous recombination (HR). An important role of Rad52 in HR is the loading of Rad51 onto replication protein A-coated single-stranded DNA (ssDNA), which is referred to as the recombination mediator activity. In vitro, Rad52 displays additional activities, including self-association, DNA binding and ssDNA annealing. Although Rad52 has been a subject of extensive genetic, biochemical and structural studies, the mechanisms by which these activities are coordinated in the various roles of Rad52 in HR remain largely unknown. In the present study, we found that an isolated C-terminal half of Rad52 disrupted the Rad51 oligomer and formed a heterodimeric complex with Rad51. The Rad52 fragment inhibited the binding of Rad51 to double-stranded DNA, but not to ssDNA. The phenylalanine-349 and tyrosine-409 residues present in the C-terminal half of Rad52 were critical for the interaction with Rad51, the disruption of Rad51 oligomers, the mediator activity of the full-length protein and for DNA repair in vivo in the presence of methyl methanesulfonate. Our studies suggested that phenylalanine-349 and tyrosine-409 are key residues in the C-terminal half of Rad52 and probably play an important role in the mediator activity.


Assuntos
Recombinação Homóloga , Proteína Rad52 de Recombinação e Reparo de DNA/química , Proteínas de Saccharomyces cerevisiae/química , Sequência de Aminoácidos , DNA/metabolismo , Reparo do DNA , Dados de Sequência Molecular , Mutação , Fenilalanina/genética , Multimerização Proteica , Rad51 Recombinase/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Tirosina/genética
8.
J Biol Chem ; 286(20): 17607-17, 2011 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-21454474

RESUMO

RecA/Rad51 proteins are essential in homologous DNA recombination and catalyze the ATP-dependent formation of D-loops from a single-stranded DNA and an internal homologous sequence in a double-stranded DNA. RecA and Rad51 require a "recombination mediator" to overcome the interference imposed by the prior binding of single-stranded binding protein/replication protein A to the single-stranded DNA. Rad52 is the prototype of recombination mediators, and the human Rad52 protein has two distinct DNA-binding sites: the first site binds to single-stranded DNA, and the second site binds to either double- or single-stranded DNA. We previously showed that yeast Rad52 extensively stimulates Rad51-catalyzed D-loop formation even in the absence of replication protein A, by forming a 2:1 stoichiometric complex with Rad51. However, the precise roles of Rad52 and Rad51 within the complex are unknown. In the present study, we constructed yeast Rad52 mutants in which the amino acid residues corresponding to the second DNA-binding site of the human Rad52 protein were replaced with either alanine or aspartic acid. We found that the second DNA-binding site is important for the yeast Rad52 function in vivo. Rad51-Rad52 complexes consisting of these Rad52 mutants were defective in promoting the formation of D-loops, and the ability of the complex to associate with double-stranded DNA was specifically impaired. Our studies suggest that Rad52 within the complex associates with double-stranded DNA to assist Rad51-mediated homologous pairing.


Assuntos
DNA Fúngico/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Recombinação Genética/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sítios de Ligação , DNA Fúngico/genética , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos
10.
J Biol Chem ; 280(37): 32218-29, 2005 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-16033757

RESUMO

Both Rad51 and Rad52 are required for homologous genetic recombination in Saccharomyces cerevisiae. Rad51 promotes heteroduplex joint formation, a general step in homologous recombination. Rad52 facilitates the binding of Rad51 to replication protein A (RPA)-coated single-stranded DNA. The requirement of RPA can be avoided in vitro, if the single-stranded DNA is short. Using short single-stranded DNA and homologous double-stranded DNA, in the absence of RPA, we found that Rad52 (optimal at three per Rad51) was still required for Rad51-promoted heteroduplex joint formation in vitro, as assayed by the formation of D-loops, suggesting another role for Rad52. Rad51 has to bind to the single-stranded DNA before the addition of double-stranded DNA for efficient D-loop formation. Immunoprecipitation and single-stranded DNA-bead precipitation analyses revealed the presence of the free and DNA-bound complexes of Rad51 and Rad52 at a 1 to 2 stoichiometry. In the presence of single-stranded DNA, in addition to Rad51, Rad52 was required for extensive untwisting that is an intermediate step toward D-loop formation. Thus, these results suggest that the formation of the stoichiometric complex of Rad52 with Rad51 on single-stranded DNA is required for the functional binding of the protein-single-stranded DNA complex to the double-stranded DNA to form D-loops.


Assuntos
Proteínas de Ligação a DNA/química , Saccharomyces cerevisiae/metabolismo , Bioquímica/métodos , Soluções Tampão , Clonagem Molecular , DNA/química , DNA/metabolismo , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Proteínas Fúngicas/química , Imunoprecipitação , Meiose , Mitose , Conformação de Ácido Nucleico , Plasmídeos/metabolismo , Ligação Proteica , Rad51 Recombinase , Proteína Rad52 de Recombinação e Reparo de DNA , Recombinases Rec A/metabolismo , Recombinação Genética , Proteínas de Saccharomyces cerevisiae , Fatores de Tempo
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