RESUMEN
DNA double-strand breaks (DSBs) threaten genome stability throughout life and are linked to tumorigenesis in humans. To initiate DSB repair by end joining or homologous recombination, the Mre11-nuclease Rad50-ATPase complex detects and processes diverse and obstructed DNA ends, but a structural mechanism is still lacking. Here we report cryo-EM structures of the E. coli Mre11-Rad50 homolog SbcCD in resting and DNA-bound cutting states. In the resting state, Mre11's nuclease is blocked by ATP-Rad50, and the Rad50 coiled coils appear flexible. Upon DNA binding, the two coiled coils zip up into a rod and, together with the Rad50 nucleotide-binding domains, form a clamp around dsDNA. Mre11 moves to the side of Rad50, binds the DNA end, and assembles a DNA cutting channel for the nuclease reactions. The structures reveal how Mre11-Rad50 can detect and process diverse DNA ends and uncover a clamping and gating function for the coiled coils.
Asunto(s)
Ácido Anhídrido Hidrolasas/metabolismo , Roturas del ADN de Doble Cadena , Replicación del ADN , ADN Bacteriano/metabolismo , Desoxirribonucleasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Exonucleasas/metabolismo , Proteína Homóloga de MRE11/metabolismo , Ácido Anhídrido Hidrolasas/genética , Ácido Anhídrido Hidrolasas/ultraestructura , Microscopía por Crioelectrón , ADN Bacteriano/genética , ADN Bacteriano/ultraestructura , Desoxirribonucleasas/genética , Desoxirribonucleasas/ultraestructura , Escherichia coli/genética , Escherichia coli/ultraestructura , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/ultraestructura , Exonucleasas/genética , Exonucleasas/ultraestructura , Proteína Homóloga de MRE11/genética , Proteína Homóloga de MRE11/ultraestructura , Conformación de Ácido Nucleico , Relación Estructura-ActividadRESUMEN
Overcoming lysogenization defect (OLD) proteins constitute a family of uncharacterized nucleases present in bacteria, archaea, and some viruses. These enzymes contain an N-terminal ATPase domain and a C-terminal Toprim domain common amongst replication, recombination, and repair proteins. The in vivo activities of OLD proteins remain poorly understood and no definitive structural information exists. Here we identify and define two classes of OLD proteins based on differences in gene neighborhood and amino acid sequence conservation and present the crystal structures of the catalytic C-terminal regions from the Burkholderia pseudomallei and Xanthamonas campestris p.v. campestris Class 2 OLD proteins at 2.24 Å and 1.86 Å resolution respectively. The structures reveal a two-domain architecture containing a Toprim domain with altered architecture and a unique helical domain. Conserved side chains contributed by both domains coordinate two bound magnesium ions in the active site of B. pseudomallei OLD in a geometry that supports a two-metal catalysis mechanism for cleavage. The spatial organization of these domains additionally suggests a novel mode of DNA binding that is distinct from other Toprim containing proteins. Together, these findings define the fundamental structural properties of the OLD family catalytic core and the underlying mechanism controlling nuclease activity.