RESUMEN
Deinococcus radiodurans (Dr) has one of the most robust DNA repair systems, which is capable of withstanding extreme doses of ionizing radiation and other sources of DNA damage. DrRecA, a central enzyme of recombinational DNA repair, is essential for extreme radioresistance. In the presence of ATP, DrRecA forms nucleoprotein filaments on DNA, similar to other bacterial RecA and eukaryotic DNA strand exchange proteins. However, DrRecA catalyzes DNA strand exchange in a unique reverse pathway. Here, we study the dynamics of DrRecA filaments formed on individual molecules of duplex and single-stranded DNA, and we follow conformational transitions triggered by ATP hydrolysis. Our results reveal that ATP hydrolysis promotes rapid DrRecA dissociation from duplex DNA, whereas on single-stranded DNA, DrRecA filaments interconvert between stretched and compressed conformations, which is a behavior shared by E. coli RecA and human Rad51. This indicates a high conservation of conformational switching in nucleoprotein filaments and suggests that additional factors might contribute to an inverse pathway of DrRecA strand exchange.
Asunto(s)
Adenosina Trifosfato/química , Proteínas Bacterianas/química , Deinococcus/enzimología , Modelos Moleculares , Conformación Molecular , Rec A Recombinasas/química , Imagen Individual de Molécula , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/metabolismo , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Hidrólisis , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Rec A Recombinasas/metabolismo , Imagen Individual de Molécula/métodosRESUMEN
The RecA protein plays a key role in bacterial homologous recombination (HR) and acts through assembly of long helical filaments around single-stranded DNA in the presence of ATP. Large-scale conformational changes induced by ATP hydrolysis result in transitions between stretched and compressed forms of the filament. Here, using a single-molecule approach, we show that compressed RecA nucleoprotein filaments can exist in two distinct interconvertible states depending on the presence of ADP in the monomer-monomer interface. Binding of ADP promotes cooperative conformational transitions and directly affects mechanical properties of the filament. Our findings reveal that RecA nucleoprotein filaments are able to continuously cycle between three mechanically distinct states that might have important implications for RecA-mediated processes of HR.