RESUMEN
Rad51 is a core component of the eukaryotic homologous recombination machinery and is responsible for key mechanistic steps during strand invasion. Higher order oligomers of Rad51 display a remarkable degree of structural variation, forming rings, compressed filaments, and elongated filaments. It is unclear whether Rad51 can transition directly between these different oligomeric structures without disassembling first into monomers. We have used single-molecule microscopy to investigate the behavior of human Rad51 assembled on double-stranded DNA. Our results show that human Rad51 can form elongated nucleoprotein filaments on DNA, but ATP hydrolysis causes a decrease in their length without concomitant dissociation of protein. Compressed Rad51 filaments can re-elongate when presented with either ATP or the non-hydrolyzable analog AMP-PNP, and these cycles of elongation and compression are reversible. A Rad51 mutant deficient in ATP hydrolysis is locked into an extended conformation that is incapable of transitioning to a compressed filament. Similarly, wild-type Rad51 bound to DNA in the presence of AMP-PNP was trapped in the elongated state. Proteins incapable of transitioning to the compressed state were also highly resistant to dissociation from the DNA. Taken together, our results indicate that nucleotide hydrolysis by human Rad51 triggers a reversible structural transition leading to filaments with reduced helical pitch.
Asunto(s)
Recombinasa Rad51/química , Adenosina Trifosfato/química , Calcio/fisiología , ADN/química , Humanos , Hidrólisis , Microscopía Fluorescente , Conformación Proteica , Cloruro de Sodio/farmacologíaRESUMEN
Rad51 is the core component of the eukaryotic homologous recombination machinery and assembles into elongated nucleoprotein filaments on DNA. We have used total internal reflection fluorescence microscopy and a DNA curtain assay to investigate the dynamics of individual Saccharomyces cerevisiae Rad51 nucleoprotein filaments. For these experiments the DNA molecules were end-labeled with single fluorescent semiconducting nanocrystals. The assembly and disassembly of the Rad51 nucleoprotein filaments were visualized by tracking the location of the labeled DNA end in real time. Using this approach, we have analyzed yeast Rad51 under a variety of different reaction conditions to assess parameters that impact the stability of the nucleoprotein filament. We show that Rad51 readily dissociates from DNA in the presence of ADP or in the absence of nucleotide cofactor, but that free ATP in solution confers a fivefold increase in the stability of the nucleoprotein filaments. We also probe how protein dissociation is coupled to ATP binding and hydrolysis by examining the effects of ATP concentration, and by the use of the nonhydrolyzable ATP analogue adenosine 5'-(beta, gamma-imido) triphosphate and ATPase active-site mutants. Finally, we demonstrate that the Rad51 gain-of-function mutant I345T dissociates from DNA with kinetics nearly identical to that of wild-type Rad51, but assembles 30% more rapidly. Together, these results provide a framework for studying the biochemical behaviors of S. cerevisiae Rad51 nucleoprotein filaments at the single-molecule level.
Asunto(s)
Nucleoproteínas , Recombinasa Rad51 , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Adenilil Imidodifosfato/metabolismo , Animales , ADN/química , ADN/metabolismo , Humanos , Cloruro de Magnesio/química , Microscopía Fluorescente/métodos , Nanopartículas , Nucleoproteínas/metabolismo , Nucleoproteínas/ultraestructura , Nucleótidos/metabolismo , Puntos Cuánticos , Recombinasa Rad51/metabolismo , Recombinasa Rad51/ultraestructura , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestructuraRESUMEN
Genetic studies in budding and fission yeasts have provided evidence that Rdh54, a Swi2/Snf2-like factor, synergizes with the Dmc1 recombinase to mediate inter-homologue recombination during meiosis. Rdh54 associates with Dmc1 in the yeast two-hybrid assay, but whether the Rdh54-Dmc1 interaction is direct and the manner in which these two recombination factors may functionally co-operate to accomplish their biological task have not yet been defined. Here, using purified Schizosaccharomyces pombe proteins, we demonstrate complex formation between Rdh54 and Dmc1 and enhancement of the recombinase activity of Dmc1 by Rdh54. Consistent with published cytological and chromatin immunoprecipitation data that implicate Rdh54 in preventing the non-specific association of Dmc1 with chromatin, we show here that Rdh54 mediates the efficient removal of Dmc1 from dsDNA. These functional attributes of Rdh54 are reliant on its ATPase function. The results presented herein provide valuable information concerning the Rdh54-Dmc1 protein pair that is germane for understanding their role in meiotic recombination. The biochemical systems established in this study should be useful for the continuing dissection of the action mechanism of Rdh54 and Dmc1.
Asunto(s)
Meiosis , Recombinasas/metabolismo , Recombinación Genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/enzimología , Adenosina Trifosfatasas/metabolismo , ADN de Hongos/metabolismo , Conformación de Ácido Nucleico , Unión Proteica , Recombinasas/aislamiento & purificación , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/aislamiento & purificaciónRESUMEN
The jaws of the bloodworm Glycera dibranchiata consist principally of protein and melanin scaffolds with small amounts of unmineralized copper (Cu) and mineralized atacamite (Cu(2)Cl(OH)(3)) fibers in distinct regions. Remarkably, when tested in air, the regions containing unmineralized Cu are the hardest, stiffest, and most abrasion resistant. To establish the functions of jaw constituents in physiologically relevant environments, this study examines the effects of hydration on their response to indentation, scratching, and wear. Although all jaw regions are degraded by the presence of water, the ones containing unmineralized Cu are affected least. Notably, scratch depths in the bulk and the atacamite-containing regions double when wet, whereas the corresponding increase in the regions with unmineralized Cu is approximately 20%. The results support the view that Cu ions are involved in the formation of intermolecular coordination complexes, creating a cross-linked molecular network that is both mechanically robust and resistant to water ingress. Hydration effects are greatest during wear testing, rates of material removal in water being about three times those in air. The mechanism underlying accelerated wear is suspected to involve coupled effects of near-surface damage and enhanced water ingress, resulting in increased plasticization and susceptibility to plastic plowing.
Asunto(s)
Anélidos/fisiología , Agua Corporal/fisiología , Modelos Biológicos , Agua/metabolismo , Animales , Fenómenos Biomecánicos/métodos , Fuerza Compresiva , Simulación por Computador , Tejido Conectivo , Elasticidad , Dureza , MaxilaresRESUMEN
The tooth-like mouthparts of some animals consist of biomacromolecular scaffolds with few mineral components, making them intriguing paradigms of biostructural materials. In this study, the abrasion resistance of the jaws of one such animal, the bloodworm Glycera dibranchiata, has been evaluated by nanoindentation, nanoscratching, and wear testing. The hardest, stiffest, and most abrasion-resistant materials are found within a thin (<3 microm) surface layer near the jaw tip and a thicker (10-20 microm) subsurface layer, both rich in unmineralized Cu. These results are consistent with the supposition that Cu ions are involved in the formation of intermolecular coordination complexes between proteins, creating a highly cross-linked molecular network. The intervening layer contains aligned atacamite [Cu(2)(OH)(3)Cl] fibers and exhibits hardness and stiffness (transverse to the alignment direction) that are only slightly higher than those of the bulk material but lower than those of the two Cu-rich layers. Furthermore, the atacamite-containing layer is the least abrasion-resistant, by a factor of approximately 3, even relative to the bulk material. These observations are broadly consistent with the behavior of engineering polymer composites with hard fiber or particulate reinforcements. The alignment of fibers parallel to the jaw surface, and the fiber proximity to the surface, are both suggestive of a natural adaptation to enhance bending stiffness and strength rather than to endow the surface regions with enhanced abrasion resistance.
Asunto(s)
Materiales Biocompatibles/química , Animales , Ensayo de Materiales , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Minerales/química , Poliquetos/ultraestructuraRESUMEN
Contrary to conventional wisdom, mineralization is not the only strategy evolved for the formation of hard, stiff materials. Indeed, the sclerotized mouthparts of marine invertebrates exhibit Young's modulus and hardness approaching 10 and 1 GPa, respectively, with little to no help from mineralization. Based on biochemical analyses, three of these mouthparts, the jaws of glycerid and nereid polychaetes and a squid beak, reveal a largely organic composition dominated by glycine- and histidine-rich proteins. Despite the well-known metal ion binding by the imidazole side-chain of histidine and the suggestion that this interaction provides mechanical support in nereid jaws, there is at present no universal molecular explanation for the relationship of histidine to mechanical properties in these sclerotized structures.
Asunto(s)
Densidad Ósea/fisiología , Invertebrados/fisiología , Calcificación de Dientes/fisiología , Diente/fisiología , Animales , Elasticidad , Dureza , Biología Marina , Estrés MecánicoRESUMEN
Defining the design principles guiding the fabrication of superior biocomposite structures from an assemblage of ordinary molecules is a key goal of biomimetics. Considering their low degree of mineralization, Glycera jaws have been shown to be extraordinarily resistant to abrasion based on the metric hardness3/Young's modulus2. The jaws also exhibit an impressive chemical inertness withstanding boiling concentrated hydrochloric acid as well as boiling concentrated sodium hydroxide. A major organic component largely responsible for the chemical inertness of the jaws has been characterized using a spectrophotometric assay for melanin content, 13C solid state nuclear magnetic resonance, IR spectroscopy, and laser desorption ionization-time of flight mass spectrometry and is identified here as a melanin-like network. Although melanin is widely distributed as a pigment in tissues and other structural biomaterials, to our knowledge, Glycera jaws represent the first known integument to exploit melanin as a cohesive load- and shape-bearing material.