RESUMEN
How living systems detect the presence of genotoxic damage embedded in a million-fold excess of undamaged DNA is an unresolved question in biology. Here we have captured and structurally elucidated a base-excision DNA repair enzyme, MutM, at the stage of initial encounter with a damaged nucleobase, 8-oxoguanine (oxoG), nested within a DNA duplex. Three structures of intrahelical oxoG-encounter complexes are compared with sequence-matched structures containing a normal G base in place of an oxoG lesion. Although the protein-DNA interfaces in the matched complexes differ by only two atoms-those that distinguish oxoG from G-their pronounced structural differences indicate that MutM can detect a lesion in DNA even at the earliest stages of encounter. All-atom computer simulations show the pathway by which encounter of the enzyme with the lesion causes extrusion from the DNA duplex, and they elucidate the critical free energy difference between oxoG and G along the extrusion pathway.
Asunto(s)
Daño del ADN , Reparación del ADN , ADN-Formamidopirimidina Glicosilasa/metabolismo , Geobacillus stearothermophilus/enzimología , Guanina/análogos & derivados , Biocatálisis , Simulación por Computador , Cristalografía por Rayos X , ADN-Formamidopirimidina Glicosilasa/genética , Genoma Bacteriano/genética , Geobacillus stearothermophilus/genética , Guanina/metabolismo , Modelos Biológicos , Modelos Moleculares , Simulación de Dinámica Molecular , Mutación/genética , TermodinámicaRESUMEN
Base excision repair of genotoxic nucleobase lesions in the genome is critically dependent upon the ability of DNA glycosylases to locate rare sites of damage embedded in a vast excess of undamaged DNA, using only thermal energy to fuel the search process. Considerable interest surrounds the question of how DNA glycosylases translocate efficiently along DNA while maintaining their vigilance for target damaged sites. Here, we report the observation of strandwise translocation of 8-oxoguanine DNA glycosylase, MutM, along undamaged DNA. In these complexes, the protein is observed to translocate by one nucleotide on one strand while remaining untranslocated on the complementary strand. We further report that alterations of single base-pairs or a single amino acid substitution (R112A) can induce strandwise translocation. Molecular dynamics simulations confirm that MutM can translocate along DNA in a strandwise fashion. These observations reveal a previously unobserved mode of movement for a DNA-binding protein along the surface of DNA.
Asunto(s)
Reparación del ADN/fisiología , ADN-Formamidopirimidina Glicosilasa/metabolismo , ADN/metabolismo , Geobacillus stearothermophilus/enzimología , Modelos Moleculares , Translocación Genética/fisiología , Cristalización , ADN-Formamidopirimidina Glicosilasa/química , ADN-Formamidopirimidina Glicosilasa/genética , Escherichia coli , Geobacillus stearothermophilus/genética , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Conformación Proteica , Transporte de Proteínas/fisiología , Sincrotrones , Difracción de Rayos XRESUMEN
MutM, a bacterial DNA glycosylase, protects genome integrity by catalyzing glycosidic bond cleavage of 8-oxoguanine (oxoG) lesions, thereby initiating base excision DNA repair. The process of searching for and locating oxoG lesions is especially challenging, because of the close structural resemblance of oxoG to its million-fold more abundant progenitor, G. Extrusion of the target nucleobase from the DNA double helix to an extrahelical position is an essential step in lesion recognition and catalysis by MutM. Although the interactions between the extruded oxoG and the active site of MutM have been well characterized, little is known in structural detail regarding the interrogation of extruded normal DNA bases by MutM. Here we report the capture and structural elucidation of a complex in which MutM is attempting to present an undamaged G to its active site. The structure of this MutM-extrahelical G complex provides insights into the mechanism MutM employs to discriminate against extrahelical normal DNA bases and into the base extrusion process in general.