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1.
Hum Mol Genet ; 22(16): 3259-68, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23595882

RESUMO

We report a novel gene for a parkinsonian disorder. X-linked parkinsonism with spasticity (XPDS) presents either as typical adult onset Parkinson's disease or earlier onset spasticity followed by parkinsonism. We previously mapped the XPDS gene to a 28 Mb region on Xp11.2-X13.3. Exome sequencing of one affected individual identified five rare variants in this region, of which none was missense, nonsense or frame shift. Using patient-derived cells, we tested the effect of these variants on expression/splicing of the relevant genes. A synonymous variant in ATP6AP2, c.345C>T (p.S115S), markedly increased exon 4 skipping, resulting in the overexpression of a minor splice isoform that produces a protein with internal deletion of 32 amino acids in up to 50% of the total pool, with concomitant reduction of isoforms containing exon 4. ATP6AP2 is an essential accessory component of the vacuolar ATPase required for lysosomal degradative functions and autophagy, a pathway frequently affected in Parkinson's disease. Reduction of the full-size ATP6AP2 transcript in XPDS cells and decreased level of ATP6AP2 protein in XPDS brain may compromise V-ATPase function, as seen with siRNA knockdown in HEK293 cells, and may ultimately be responsible for the pathology. Another synonymous mutation in the same exon, c.321C>T (p.D107D), has a similar molecular defect of exon inclusion and causes X-linked mental retardation Hedera type (MRXSH). Mutations in XPDS and MRXSH alter binding sites for different splicing factors, which may explain the marked differences in age of onset and manifestations.


Assuntos
Cromossomos Humanos X , Doenças Genéticas Ligadas ao Cromossomo X/genética , Variação Genética , Espasticidade Muscular/genética , Transtornos Parkinsonianos/genética , Receptores de Superfície Celular/genética , ATPases Vacuolares Próton-Translocadoras/genética , Idoso , Sítios de Ligação/genética , Células Cultivadas , Códon sem Sentido , Exoma , Feminino , Mutação da Fase de Leitura , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Ligação Genética , Células HEK293 , Humanos , Masculino , Deficiência Intelectual Ligada ao Cromossomo X/genética , Deficiência Intelectual Ligada ao Cromossomo X/metabolismo , Espasticidade Muscular/metabolismo , Mutação de Sentido Incorreto , Transtornos Parkinsonianos/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Análise de Sequência de RNA , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/metabolismo
2.
Nucleic Acids Res ; 35(12): 4124-40, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17567608

RESUMO

Rad51 and Rad54 are key proteins that collaborate during homologous recombination. Rad51 forms a presynaptic filament with ATP and ssDNA active in homology search and DNA strand exchange, but the precise role of its ATPase activity is poorly understood. Rad54 is an ATP-dependent dsDNA motor protein that can dissociate Rad51 from dsDNA, the product complex of DNA strand exchange. Kinetic analysis of the budding yeast proteins revealed that the catalytic efficiency of the Rad54 ATPase was stimulated by partial filaments of wild-type and Rad51-K191R mutant protein on dsDNA, unambiguously demonstrating that the Rad54 ATPase activity is stimulated under these conditions. Experiments with Rad51-K191R as well as with wild-type Rad51-dsDNA filaments formed in the presence of ATP, ADP or ATP-gamma-S showed that efficient Rad51 turnover from dsDNA requires both the Rad51 ATPase and the Rad54 ATPase activities. The results with Rad51-K191R mutant protein also revealed an unexpected defect in binding to DNA. Once formed, Rad51-K191R-DNA filaments appeared normal upon electron microscopic inspection, but displayed significantly increased stability. These biochemical defects in the Rad51-K191R protein could lead to deficiencies in presynapsis (filament formation) and postsynapsis (filament disassembly) in vivo.


Assuntos
Adenosina Trifosfatases/metabolismo , DNA/metabolismo , Rad51 Recombinase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo , Substituição de Aminoácidos , DNA/química , DNA/ultraestrutura , DNA Helicases , Enzimas Reparadoras do DNA , DNA Super-Helicoidal/análise , Rad51 Recombinase/genética , Rad51 Recombinase/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação
3.
Proc Natl Acad Sci U S A ; 103(26): 9767-72, 2006 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-16785421

RESUMO

Rad54 protein is a Snf2-related dsDNA-specific ATPase essential for homologous recombination mediated by Rad51 protein, the eukaryotic RecA ortholog. Snf2-related enzymes couple ATP hydrolysis with translocation on dsDNA to remodel or dissociate a wide variety of protein-dsDNA complexes. Rad54 and Rad51 interact through species-specific contacts and mutually stimulate their biochemical activities. Specifically, Rad51 bound to dsDNA, the product of homologous recombination after DNA-strand exchange, stimulates the Rad54 ATPase up to 6-fold, leading to the turnover of Rad51 in the product complex. Electron microscopy visualized the Rad51-Rad54 interaction on dsDNA, showing that an oligomeric form of Rad54 associates preferentially with termini of the Rad51-dsDNA filament. Our data support a mechanism of processive dsDNA-Rad51 filament dissociation by the translocating Rad54 protein.


Assuntos
Rad51 Recombinase/química , Proteínas de Saccharomyces cerevisiae/química , Adenosina Trifosfatases , DNA/química , DNA/metabolismo , DNA Helicases , Enzimas Reparadoras do DNA , Proteínas de Ligação a DNA/química , Microscopia Eletrônica , Mapeamento de Interação de Proteínas , Transporte Proteico , Rad51 Recombinase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/química
4.
J Biol Chem ; 280(28): 26303-11, 2005 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-15908697

RESUMO

Rad51 is a homolog of the bacterial RecA protein and is central for recombination in eukaryotes performing homology search and DNA strand exchange. Rad51 and RecA share a core ATPase domain that is structurally similar to the ATPase domains of helicases and the F1 ATPase. Rad51 has an additional N-terminal domain, whereas RecA protein has an additional C-terminal domain. Here we show that glycine 103 in the N-terminal domain of Saccharomyces cerevisiae Rad51 is important for binding to single-stranded and duplex DNA. The Rad51-G103E mutant protein is deficient in DNA strand exchange and ATPase activity due to a primary DNA binding defect. The N-terminal domain of Rad51 is connected to the ATPase core through an extended elbow linker that ensures flexibility of the N-terminal domain. Molecular modeling of the Rad51-G103E mutant protein shows that the negatively charged glutamate residue lies on the surface of the N-terminal domain facing a positively charged patch composed of Arg-260, His-302, and Lys-305 on the ATPase core domain. A possible structural explanation for the DNA binding defect is that a charge interaction between Glu-103 and the positive patch restricts the flexibility of the N-terminal domain. Rad51-G103E was identified in a screen for Rad51 interaction-deficient mutants and was shown to ablate the Rad54 interaction in two-hybrid assays (Krejci, L., Damborsky, J., Thomsen, B., Duno, M., and Bendixen, C. (2001) Mol. Cell. Biol. 21, 966-976). Surprisingly, we found that the physical interaction of Rad51-G103E with Rad54 was not affected. Our data suggest that the two-hybrid interaction defect was an indirect consequence of the DNA binding defect.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Glicina/química , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/química , Trifosfato de Adenosina/química , Sequência de Aminoácidos , Arginina/química , DNA/química , DNA Helicases , Enzimas Reparadoras do DNA , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Ácido Glutâmico/química , Glutationa Transferase/metabolismo , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Rad51 Recombinase , Recombinases Rec A/química , Proteínas de Saccharomyces cerevisiae/química , Homologia de Sequência de Aminoácidos , Cloreto de Sódio/farmacologia , Software , Técnicas do Sistema de Duplo-Híbrido
5.
Mol Cell ; 10(5): 1175-88, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12453424

RESUMO

Rad54 protein is a member of the Swi2/Snf2-like family of DNA-dependent/stimulated ATPases that dissociate and remodel protein complexes on dsDNA. Rad54 functions in the recombinational DNA repair (RAD52) pathway. Here we show that Rad54 protein dissociates Rad51 from nucleoprotein filaments formed on dsDNA. Addition of Rad54 protein overcomes inhibition of DNA strand exchange by Rad51 protein bound to substrate dsDNA. Species preference in the Rad51 dissociation and DNA strand exchange assays underlines the importance of specific Rad54-Rad51 protein interactions. Rad51 protein is unable to release dsDNA upon ATP hydrolysis, leaving it stuck on the heteroduplex DNA product after DNA strand exchange. We suggest that Rad54 protein is involved in the turnover of Rad51-dsDNA filaments.


Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , DNA/química , Proteínas Fúngicas/metabolismo , Proteínas Nucleares , Recombinação Genética , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , DNA/metabolismo , DNA Helicases , Enzimas Reparadoras do DNA , Proteínas de Ligação a DNA/química , Proteínas Fúngicas/química , Ligação Proteica , Rad51 Recombinase , Saccharomyces cerevisiae/metabolismo , Cloreto de Sódio/farmacologia , Fatores de Tempo
6.
J Biol Chem ; 277(48): 46205-15, 2002 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-12359723

RESUMO

Rad54 protein is a Snf2-like ATPase with a specialized function in the recombinational repair of DNA damage. Rad54 is thought to stimulate the search of homology via formation of a specific complex with the presynaptic Rad51 filament on single-stranded DNA. Herein, we address the interaction of Rad54 with Rad51 filaments on double-stranded (ds) DNA, an intermediate in DNA strand exchange with unclear functional significance. We show that Saccharomyces cerevisiae Rad54 exerts distinct modes of ATPase activity on partially and fully saturated filaments of Rad51 protein on dsDNA. The highest ATPase activity is observed on dsDNA containing short patches of yeast Rad51 filaments resulting in a 6-fold increase compared with protein-free DNA. This enhanced ATPase mode of yeast Rad54 can also be elicited by partial filaments of human Rad51 protein but to a lesser extent. In contrast, the interaction of Rad54 protein with duplex DNA fully covered with Rad51 is entirely species-specific. When yeast Rad51 fully covers dsDNA, Rad54 protein hydrolyzes ATP in a reduced mode at 60-80% of its rate on protein-free DNA. Instead, saturated filaments with human Rad51 fail to support the yeast Rad54 ATPase. We suggest that the interaction of Rad54 with dsDNA-Rad51 complexes is of functional importance in homologous recombination.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas Fúngicas/fisiologia , Proteínas de Saccharomyces cerevisiae , DNA Helicases , Enzimas Reparadoras do DNA , Proteínas Fúngicas/metabolismo , Rad51 Recombinase , Saccharomyces cerevisiae/metabolismo
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