RESUMEN
DNA mismatch repair detects and removes mismatches from DNA by a conserved mechanism, reducing the error rate of DNA replication by 100- to 1,000-fold. In this process, MutS homologs scan DNA, recognize mismatches and initiate repair. How the MutS homologs selectively license repair of a mismatch among millions of matched base pairs is not understood. Here we present four cryo-EM structures of Escherichia coli MutS that provide snapshots, from scanning homoduplex DNA to mismatch binding and MutL activation via an intermediate state. During scanning, the homoduplex DNA forms a steric block that prevents MutS from transitioning into the MutL-bound clamp state, which can only be overcome through kinking of the DNA at a mismatch. Structural asymmetry in all four structures indicates a division of labor between the two MutS monomers. Together, these structures reveal how a small conformational change from the homoduplex- to heteroduplex-bound MutS acts as a licensing step that triggers a dramatic conformational change that enables MutL binding and initiation of the repair cascade.
Asunto(s)
ADN/ultraestructura , Proteínas de Escherichia coli/ultraestructura , Proteínas MutL/ultraestructura , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/ultraestructura , Conformación Proteica , Microscopía por Crioelectrón , ADN/genética , Reparación de la Incompatibilidad de ADN/genética , Reparación del ADN/genética , Replicación del ADN/genética , Escherichia coli/genética , Escherichia coli/ultraestructura , Proteínas de Escherichia coli/genética , Proteínas MutL/genética , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/genéticaRESUMEN
DNA mismatch repair (MMR) corrects mismatches, small insertions and deletions in DNA during DNA replication. While scanning for mismatches, dimers of MutS embrace the DNA helix with their lever and clamp domains. Previous studies indicated generic flexibility of the lever and clamp domains of MutS prior to DNA binding, but whether this was important for MutS function was unknown. Here, we present a novel crystal structure of DNA-free Escherichia coli MutS. In this apo-structure, the clamp domains are repositioned due to kinking at specific sites in the coiled-coil region in the lever domains, suggesting a defined hinge point. We made mutations at the coiled-coil hinge point. The mutants made to disrupt the helical fold at the kink site diminish DNA binding, whereas those made to increase stability of coiled-coil result in stronger DNA binding. These data suggest that the site-specific kinking of the coiled-coil in the lever domain is important for loading of this ABC-ATPase on DNA.