RESUMEN
SWI2/SNF2 chromatin-remodeling proteins mediate the mobilization of nucleosomes and other DNA-associated proteins. SWI2/SNF2 proteins contain sequence motifs characteristic of SF2 helicases but do not have helicase activity. Instead, they couple ATP hydrolysis with the generation of superhelical torsion in DNA. The structure of the nucleosome-remodeling domain of zebrafish Rad54, a protein involved in Rad51-mediated homologous recombination, reveals that the core of the SWI2/SNF2 enzymes consist of two alpha/beta-lobes similar to SF2 helicases. The Rad54 helicase lobes contain insertions that form two helical domains, one within each lobe. These insertions contain SWI2/SNF2-specific sequence motifs likely to be central to SWI2/SNF2 function. A broad cleft formed by the two lobes and flanked by the helical insertions contains residues conserved in SWI2/SNF2 proteins and motifs implicated in DNA-binding by SF2 helicases. The Rad54 structure suggests that SWI2/SNF2 proteins use a mechanism analogous to helicases to translocate on dsDNA.
Asunto(s)
Cromatina/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Línea Celular , Cristalografía , ADN Helicasas/química , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Alineación de Secuencia , Factores de Transcripción/química , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Homologous recombination is important for the repair of double-stranded DNA breaks in all organisms. Rad51 and Rad54 proteins are two key components of the homologous recombination machinery in eukaryotes. In vitro, Rad51 protein assembles with single-stranded DNA to form the helical nucleoprotein filament that promotes DNA strand exchange, a basic step of homologous recombination. Rad54 protein interacts with this Rad51 nucleoprotein filament and stimulates its DNA pairing activity, suggesting that Rad54 protein is a component of the nucleoprotein complex involved in the DNA homology search. Here, using physical criteria, we demonstrate directly the formation of Rad54-Rad51-DNA nucleoprotein co-complexes that contain equimolar amounts of each protein. The binding of Rad54 protein significantly stabilizes the Rad51 nucleoprotein filament formed on either single-stranded DNA or double-stranded DNA. The Rad54-stabilized nucleoprotein filament is more competent in DNA strand exchange and acts over a broader range of solution conditions. Thus, the co-assembly of an interacting partner with the Rad51 nucleoprotein filament represents a novel means of stabilizing the biochemical entity central to homologous recombination, and reveals a new function of Rad54 protein.