RESUMEN
The tumor suppressor BRCA2 plays a key role in initiating homologous recombination by facilitating RAD51 filament formation on single-stranded DNA. The small acidic protein DSS1 is a crucial partner to BRCA2 in this process. In vitro and in cells (1,2), BRCA2 associates into oligomeric complexes besides also existing as monomers. A dimeric structure was further characterized by electron microscopic analysis (3), but the functional significance of the different BRCA2 assemblies remains to be determined. Here, we used biochemistry and electron microscopic imaging to demonstrate that the multimerization of BRCA2 is counteracted by DSS1 and ssDNA. When validating the findings, we identified three self-interacting regions and two types of self-association, the N-to-C terminal and the N-to-N terminal interactions. The N-to-C terminal self-interaction of BRCA2 is sensitive to DSS1 and ssDNA. The N-to-N terminal self-interaction is modulated by ssDNA. Our results define a novel role of DSS1 to regulate BRCA2 in an RPA-independent fashion. Since DSS1 is required for BRCA2 function in recombination, we speculate that the monomeric and oligomeric forms of BRCA2 might be active for different cellular events in recombinational DNA repair and replication fork stabilization.
Asunto(s)
Proteína BRCA2/metabolismo , ADN de Cadena Simple/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Proteína BRCA2/química , Proteína BRCA2/genética , Proteína BRCA2/ultraestructura , Línea Celular , Cricetulus , Humanos , Multimerización de ProteínaRESUMEN
In the 1960s, I developed methods for directly visualizing DNA and DNA-protein complexes using an electron microscope. This made it possible to examine the shape of DNA and to visualize proteins as they fold and loop DNA. Early applications included the first visualization of true nucleosomes and linkers and the demonstration that repeating tracts of adenines can cause a curvature in DNA. The binding of DNA repair proteins, including p53 and BRCA2, has been visualized at three- and four-way junctions in DNA. The trombone model of DNA replication was directly verified, and the looping of DNA at telomeres was discovered.
Asunto(s)
Proteínas de Unión al ADN/ultraestructura , ADN/ultraestructura , Microscopía Electrónica/métodos , Nucleosomas/ultraestructura , Proteína BRCA2/metabolismo , Proteína BRCA2/ultraestructura , ADN/química , ADN/metabolismo , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Conformación de Ácido Nucleico , Nucleosomas/genética , Nucleosomas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/ultraestructuraRESUMEN
Individuals with BRCA2 mutations are predisposed to breast cancers owing to genome instability. To determine the functions of BRCA2, the human protein was purified. It was found to bind selectively to single-stranded DNA (ssDNA), and to ssDNA in tailed duplexes and replication fork structures. Monomeric and dimeric forms of BRCA2 were observed by EM. BRCA2 directed the binding of RAD51 recombinase to ssDNA, reduced the binding of RAD51 to duplex DNA and stimulated RAD51-mediated DNA strand exchange. These observations provide a molecular basis for the role of BRCA2 in the maintenance of genome stability.