RESUMO
Intercellular communication is an essential process in all multicellular organisms. During this process, molecules secreted by one cell will bind to a receptor on the cognate cell leading to the subsequent uptake of the receptor-ligand complex. Once inside, the cell then determines the fate of the receptor-ligand complex and any other proteins that were endocytosed together. Approximately 80% of endocytosed material is recycled back to the plasma membrane either directly or indirectly via the Golgi apparatus and the remaining 20% is delivered to the lysosome for degradation. Although most pathways have been identified, we still lack understanding on how specificity in sorting of recycling cargos into different pathways is achieved, and how the cell reaches high accuracy of these processes in the absence of clear sorting signals in the bulk of the client proteins. In this review, we will summarize our current understanding of the mechanism behind recycling cargo sorting and propose a model of differential affinities between cargo and cargo receptors/adaptors with regards to iterative sorting in endosomes.
Assuntos
Endocitose , Endossomos , Humanos , Ligantes , Endossomos/metabolismo , Transporte Proteico , Proteínas/metabolismo , Comunicação CelularRESUMO
Post-translational modifications are key in the regulation of activity, structure, localization, and stability of most proteins in eukaryotes. Phosphorylation is potentially the most studied post-translational modification, also due to its reversibility and thereby the regulatory role this modification often plays. While most research attention was focused on kinases in the past, phosphatases remain understudied, most probably because the addition and presence of the modification is more easily studied than its removal and absence. Here, we report the identification of an uncharacterized protein tyrosine phosphatase PPH-7 in C. elegans, a member of the evolutionary conserved PTPN family of phosphatases. Lack of PPH-7 function led to reduction of fertility and embryonic lethality at elevated temperatures. Proteomics revealed changes in the regulation of targets of the von Hippel-Lindau (VHL) E3 ligase, suggesting a potential role for PPH-7 in the regulation of VHL.
Assuntos
Caenorhabditis elegans , Proteína Supressora de Tumor Von Hippel-Lindau , Animais , Caenorhabditis elegans/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Temperatura , Proteínas Tirosina Fosfatases , Desenvolvimento Embrionário/genética , Fertilidade/genéticaRESUMO
Although acetylated alpha-tubulin is known to be a marker of stable microtubules in neurons, precise factors that regulate alpha-tubulin acetylation are, to date, largely unknown. Therefore, a genetic screen was employed in the nematode Caenorhabditis elegans that identified the Elongator complex as a possible regulator of alpha-tubulin acetylation. Detailed characterization of mutant animals revealed that the acetyltransferase activity of the Elongator is indeed required for correct acetylation of microtubules and for neuronal development. Moreover, the velocity of vesicles on microtubules was affected by mutations in Elongator. Elongator mutants also displayed defects in neurotransmitter levels. Furthermore, acetylation of alpha-tubulin was shown to act as a novel signal for the fine-tuning of microtubules dynamics by modulating alpha-tubulin turnover, which in turn affected neuronal shape. Given that mutations in the acetyltransferase subunit of the Elongator (Elp3) and in a scaffold subunit (Elp1) have previously been linked to human neurodegenerative diseases, namely Amyotrophic Lateral Sclerosis and Familial Dysautonomia respectively highlights the importance of this work and offers new insights to understand their etiology.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Transporte/metabolismo , Histona Acetiltransferases/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Tubulina (Proteína)/metabolismo , Acetilação , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Transporte/genética , Histona Acetiltransferases/genética , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Proteínas de Ligação a RNA , Tubulina (Proteína)/genéticaRESUMO
Cellular organization, compartmentalization and cell-to-cell communication are crucially dependent on endosomal pathways. Sorting endosomes provide a transit point for various trafficking pathways and decide the fate of proteins: recycling, secretion or degradation. FERARI (Factors for Endosome Recycling and Rab Interactions) play a key role in shaping these compartments and coordinate Rab GTPase function with membrane fusion and fission of vesicles through a kiss-and-run mechanism. Here, we show that FERARI also mediate kiss-and-run of Rab5-positive vesicles with sorting endosomes. During these encounters, cargo flows from Rab5-positive vesicles into sorting endosomes and from there in Rab11-positive vesicles. Cargo flow from sorting endosomes into Rab11 structures relies on the cargo adaptor SNX6, while cargo retention in the Rab11 compartment is dependent on AP1. The available cargo amount appears to regulate the duration of kisses. We propose that FERARI, together with cargo adaptors, coordinate the vectorial flow of cargo through sorting endosomes.
Assuntos
Endossomos , Proteínas rab de Ligação ao GTP , Comunicação Celular , Endossomos/metabolismo , Fusão de Membrana , Transporte Proteico , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Endosomal transport is essential for cellular organization and compartmentalization and cell-cell communication. Sorting endosomes provide a crossroads for various trafficking pathways and determine recycling, secretion or degradation of proteins. The organization of these processes requires membrane-tethering factors to coordinate Rab GTPase function with membrane fusion. Here, we report a conserved tethering platform that acts in the Rab11 recycling pathways at sorting endosomes, which we name factors for endosome recycling and Rab interactions (FERARI). The Rab-binding module of FERARI consists of Rab11FIP5 and rabenosyn-5/RABS-5, while the SNARE-interacting module comprises VPS45 and VIPAS39. Unexpectedly, the membrane fission protein EHD1 is also a FERARI component. Thus, FERARI appears to combine fusion activity through the SM protein VPS45 with pinching activity through EHD1 on SNX-1-positive endosomal membranes. We propose that coordination of fusion and pinching through a kiss-and-run mechanism drives cargo at endosomes into recycling pathways.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Endocitose/genética , Endossomos/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Comunicação Celular , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Intestinos/citologia , Imagem Molecular , Oócitos/citologia , Oócitos/metabolismo , Transporte Proteico , Nexinas de Classificação/genética , Nexinas de Classificação/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
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 & purificaçãoRESUMO
The Holliday junction is a key recombination intermediate whose resolution generates crossovers. Interplay between recombination, repair and replication has moved the Holliday junction to the center stage of nuclear DNA metabolism. Holliday junction resolvases in the eukaryotic nucleus have long eluded identification. The endonucleases Mus81/Mms4-Eme1 and XPF-MEI-9/MUS312 are structurally related to the archaeal resolvase Hjc and were found to be involved in crossover formation in budding yeast and flies, respectively. Although these endonucleases might represent one class of eukaryotic resolvases, their substrate preference opens up the possibility that junctions other than classical Holliday junctions might contribute to crossovers. Holliday junction resolution to non-crossover products can also be achieved topologically, for example, by the action of RecQ-like DNA helicases combined with topoisomerase III.
Assuntos
Núcleo Celular/metabolismo , Replicação do DNA , Recombinação Genética , Alelos , Animais , Proteínas de Bactérias/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/química , Humanos , Conformação de Ácido Nucleico , Fenótipo , Especificidade por SubstratoRESUMO
Rad54 plays key roles in homologous recombination (HR) and double-strand break (DSB) repair in yeast, along with Rad51, Rad52, Rad55 and Rad57. Rad54 belongs to the Swi2/Snf2 family of DNA-stimulated ATPases. Rad51 nucleoprotein filaments catalyze DNA strand exchange and Rad54 augments this activity of Rad51. Mutations in the Rad54 ATPase domain (ATPase(-)) impair Rad54 function in vitro, sensitize yeast to killing by methylmethane sulfonate and reduce spontaneous gene conversion. We found that overexpression of ATPase(-) Rad54 reduced spontaneous direct repeat gene conversion and increased both spontaneous direct repeat deletion and spontaneous allelic conversion. Overexpression of ATPase(-) Rad54 decreased DSB-induced allelic conversion, but increased chromosome loss and DSB-dependent lethality. Thus, ATP hydrolysis by Rad54 contributes to genome stability by promoting high-fidelity DSB repair and suppressing spontaneous deletions. Overexpression of wild-type Rad54 did not alter DSB-induced HR levels, but conversion tract lengths were reduced. Interestingly, ATPase(-) Rad54 decreased overall HR levels and increased tract lengths. These tract length changes provide new in vivo evidence that Rad54 functions in the post-synaptic phase during recombinational repair of DSBs.
Assuntos
Adenosina Trifosfatases/metabolismo , Dano ao DNA , Proteínas Fúngicas/metabolismo , Conversão Gênica/genética , Recombinação Genética/genética , Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfatases/genética , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , DNA Helicases , Enzimas Reparadoras do DNA , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Genótipo , Metanossulfonato de Metila/farmacologia , Mutação , Sequências Repetitivas de Ácido Nucleico/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Deleção de SequênciaRESUMO
In eukaryotes, mutations in a number of genes that affect DNA damage checkpoints or DNA replication also affect telomere length [Curr. Opin. Cell Biol. 13 (2001) 281]. Saccharomyces cerevisae strains with mutations in the TEL1 gene (encoding an ATM-like protein kinase) have very short telomeres, as do strains with mutations in XRS2, RAD50, or MRE11 (encoding members of a trimeric complex). Xrs2p and Mre11p are phosphorylated in a Tel1p-dependent manner in response to DNA damage [Genes Dev. 15 (2001) 2238; Mol. Cell 7 (2001) 1255]. We found that Xrs2p, but not Mre11p or Rad50p, is efficiently phosphorylated in vitro by immunopreciptated Tel1p. Strains with mutations eliminating all SQ and TQ motifs in Xrs2p (preferred targets of the ATM kinase family) had wild-type length telomeres and wild-type sensitivity to DNA damaging agents. We also showed that Rfa2p (a subunit of RPA) and the Dun1p checkpoint kinase, which are required for DNA damage repair and which are phosphorylated in response to DNA damage in vivo, are in vitro substrates of the Tel1p and Mec1p kinases. In addition, Dun1p substrates with no SQ or TQ motifs are phosphorylated by Mec1p in vitro very inefficiently, but retain most of their ability to be phosphorylated by Tel1p. We demonstrated that null alleles of DUN1 and certain mutant alleles of RFA2 result in short telomeres. As observed with Xrs2p, however, strains with mutations of DUN1 or RFA2 that eliminate SQ motifs have no effect on telomere length or DNA damage sensitivity.
Assuntos
Proteínas de Ciclo Celular , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas Quinases/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Telômero/genética , Fatores de Transcrição/genética , Aminoácidos , Dano ao DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases , Exodesoxirribonucleases , Peptídeos e Proteínas de Sinalização Intracelular , Mutação , Fosforilação , Plasmídeos , Testes de Precipitina , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Proteína de Replicação A , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Transformação GenéticaRESUMO
The end of the life of a transport vesicle requires a complex series of tethering, docking, and fusion events. Tethering complexes play a crucial role in the recognition of membrane entities and bringing them into close opposition, thereby coordinating and controlling cellular trafficking events. Here we provide a comprehensive RNA interference analysis of the CORVET and HOPS tethering complexes in metazoans. Knockdown of CORVET components promoted RAB-7 recruitment to subapical membranes, whereas in HOPS knockdowns, RAB-5 was found also on membrane structures close to the cell center, indicating the RAB conversion might be impaired in the absence of these tethering complexes. Unlike in yeast, metazoans have two VPS33 homologues, which are Sec1/Munc18 (SM)-family proteins involved in the regulation of membrane fusion. We assume that in wild type, each tethering complex contains a specific SM protein but that they may be able to substitute for each other in case of absence of the other. Of importance, knockdown of both SM proteins allowed bypass of the endosome maturation block in sand-1 mutants. We propose a model in which the SM proteins in tethering complexes are required for coordinated flux of material through the endosomal system.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Endossomos/metabolismo , Proteínas Munc18/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Endocitose/genética , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Lisossomos/metabolismo , Fusão de Membrana , Microscopia de Fluorescência , Proteínas Munc18/genética , Mutação , Oócitos/metabolismo , Transporte Proteico/genética , Interferência de RNA , Vesículas Transportadoras/metabolismo , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
RNA interference (RNAi) is a powerful tool to study the intracellular membrane transport and membrane organelle behavior in the nematode Caenorhabditis elegans. This model organism has gained popularity in the trafficking field because of its relative simplicity, yet being multicellular. C. elegans is fully sequenced and has an annotated genome, it is easy to maintain, and a growing number of transgenic strains bearing markers for different membrane compartments are available. C. elegans is particularly well suited for protein downregulation by RNAi because of the simple but efficient methods of dsRNA delivery. The phenomenon of systemic RNAi in the worm further facilitates this approach. In this chapter we describe methods and applications of RNAi in the field of membrane traffic. We summarize the fluorescent markers used as a readout for the effects of gene knockdown in different cells and tissues and give details for data acquisition and analysis.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Membrana Celular/metabolismo , Proteínas Luminescentes/metabolismo , Interferência de RNA , Animais , Biomarcadores/metabolismo , Expressão Gênica , Técnicas de Silenciamento de Genes , Processamento de Imagem Assistida por Computador , Microscopia Confocal , Fenótipo , Transporte ProteicoRESUMO
Endocytosis describes the processes by which proteins, peptides and solutes, and also pathogens, enter the cell. Endocytosed material progresses to endosomes. Genetic studies in yeast, worms, flies and mammals have identified a set of universally conserved proteins that are essential for early-to-late endosome transition and lysosome biogenesis, and for endolysosomal trafficking pathways, including autophagy. The two Vps-C complexes CORVET (class C core vacuole/endosome tethering) and HOPS (homotypic fusion and vacuole protein sorting) perform diverse biochemical functions in endocytosis: they tether membranes, interact with Rab GTPases, activate and proof-read SNARE assembly to drive membrane fusion, and possibly attach endosomes to the cytoskeleton. In addition, several of the CORVET and HOPS subunits have diversified in metazoans, and probably form additional specialized complexes to accomodate the higher complexity of trafficking pathways in these cells. Recent studies offer new insights into the complex relationships between CORVET and HOPS complexes and other factors of the endolysosomal pathway. Interactions with V-ATPase, the ESCRT machinery, phosphoinositides, the cytoskeleton and the Rab switch suggest an intricate cooperative network for endosome maturation. Accumulating evidence supports the view that endosomal tethering complexes implement a regulatory logic that governs endomembrane identity and dynamics.
Assuntos
Proteínas de Transporte Vesicular/fisiologia , Animais , Endocitose , Complexos Endossomais de Distribuição Requeridos para Transporte/fisiologia , Endossomos/metabolismo , Genes Fúngicos , Humanos , Fusão de Membrana , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/fisiologia , Transporte Proteico , Vacúolos/fisiologia , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genéticaRESUMO
Small GTPases of the Sar/Arf family are essential to generate transport containers that mediate communication between organelles of the secretory pathway. Guanine nucleotide exchange factor (GEFs) activate the small GTPases and help their anchorage in the membrane. Thus, GEFs in a way temporally and spatially control Sar1/Arf1 GTPase activation. We investigated the role of the ArfGEF GBF-1 in C. elegans oocytes and intestinal epithelial cells. GBF-1 localizes to the cis-Golgi and is part of the t-ER-Golgi elements. GBF-1 is required for secretion and Golgi integrity. In addition, gbf-1(RNAi) causes the ER reticular structure to become dispersed, without destroying ER exit sites (ERES) because the ERES protein SEC-16 was still localized in distinct punctae at t-ER-Golgi units. Moreover, GBF-1 plays a role in receptor-mediated endocytosis in oocytes, without affecting recycling pathways. We find that both the yolk receptor RME-2 and the recycling endosome-associated RAB-11 localize similarly in control and gbf-1(RNAi) oocytes. While RAB5-positive early endosomes appear to be less prominent and the RAB-5 levels are reduced by gbf-1(RNAi) in the intestine, RAB-7-positive late endosomes were more abundant and formed aggregates and tubular structures. Our data suggest a role for GBF-1 in ER structure and endosomal traffic.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Retículo Endoplasmático/fisiologia , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Animais , Caenorhabditis elegans/genética , Células Cultivadas , Endocitose , Retículo Endoplasmático/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/citologia , Oócitos/metabolismo , Interferência de RNARESUMO
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/químicaRESUMO
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-HíbridoRESUMO
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 TempoRESUMO
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.