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) maintains genome stability through repair of DNA replication errors. In Escherichia coli, initiation of MMR involves recognition of the mismatch by MutS, recruitment of MutL, activation of endonuclease MutH and DNA strand incision at a hemimethylated GATC site. Here, we studied the mechanism of communication that couples mismatch recognition to daughter strand incision. We investigated the effect of catalytically-deficient Cas9 as well as stalled RNA polymerase as roadblocks placed on DNA in between the mismatch and GATC site in ensemble and single molecule nanomanipulation incision assays. The MMR proteins were observed to incise GATC sites beyond a roadblock, albeit with reduced efficiency. This residual incision is completely abolished upon shortening the disordered linker regions of MutL. These results indicate that roadblock bypass can be fully attributed to the long, disordered linker regions in MutL and establish that communication during MMR initiation occurs along the DNA backbone.
Asunto(s)
Reparación de la Incompatibilidad de ADN/genética , ADN Bacteriano/genética , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas MutL/metabolismo , Disparidad de Par Base/genética , Proteína 9 Asociada a CRISPR/genética , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , Endodesoxirribonucleasas/metabolismo , Inestabilidad Genómica/genética , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/metabolismoRESUMEN
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.
Asunto(s)
Apoproteínas/química , ADN Bacteriano/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/química , Secuencia de Aminoácidos , Apoproteínas/genética , Apoproteínas/metabolismo , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Cinética , Modelos Moleculares , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/genética , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/metabolismo , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Relación Estructura-ActividadRESUMEN
BRCA1-BARD1-catalyzed ubiquitination of histone H2A is an important regulator of the DNA damage response, priming chromatin for repair by homologous recombination. However, no specific deubiquitinating enzymes (DUBs) are known to antagonize this function. Here we identify ubiquitin specific protease-48 (USP48) as a H2A DUB, specific for the C-terminal BRCA1 ubiquitination site. Detailed biochemical analysis shows that an auxiliary ubiquitin, an additional ubiquitin that itself does not get cleaved, modulates USP48 activity, which has possible implications for its regulation in vivo. In cells we reveal that USP48 antagonizes BRCA1 E3 ligase function and in BRCA1-proficient cells loss of USP48 results in positioning 53BP1 further from the break site and in extended resection lengths. USP48 repression confers a survival benefit to cells treated with camptothecin and its activity acts to restrain gene conversion and mutagenic single-strand annealing. We propose that USP48 promotes genome stability by antagonizing BRCA1 E3 ligase function.
Asunto(s)
Proteína BRCA1/metabolismo , Histonas/metabolismo , Proteasas Ubiquitina-Específicas/metabolismo , Ubiquitina/metabolismo , Animales , Proteína BRCA1/genética , Secuencia de Bases , Línea Celular Tumoral , Células Cultivadas , Reparación del ADN , Células HeLa , Humanos , Cinética , Ratones Noqueados , Interferencia de ARN , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteasas Ubiquitina-Específicas/genética , UbiquitinaciónRESUMEN
DNA mismatch repair (MMR) is an evolutionarily-conserved process responsible for the repair of replication errors. In Escherichia coli, MMR is initiated by MutS and MutL, which activate MutH to incise transiently-hemimethylated GATC sites. MMR efficiency depends on the distribution of these GATC sites. To understand which molecular events determine repair efficiency, we quantitatively studied the effect of strand incision on unwinding and excision activity. The distance between mismatch and GATC site did not influence the strand incision rate, and an increase in the number of sites enhanced incision only to a minor extent. Two GATC sites were incised by the same activated MMR complex in a processive manner, with MutS, the closed form of MutL and MutH displaying different roles. Unwinding and strand excision were more efficient on a substrate with two nicks flanking the mismatch, as compared to substrates containing a single nick or two nicks on the same side of the mismatch. Introduction of multiple nicks by the human MutLα endonuclease also contributed to increased repair efficiency. Our data support a general model of prokaryotic and eukaryotic MMR in which, despite mechanistic differences, mismatch-activated complexes facilitate efficient repair by creating multiple daughter strand nicks.
Asunto(s)
Reparación de la Incompatibilidad de ADN , Replicación del ADN , Disparidad de Par Base/genética , Secuencia de Bases , Metilación de ADN/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Células HEK293 , Humanos , Modelos Biológicos , Conformación ProteicaRESUMEN
To avoid mutations in the genome, DNA replication is generally followed by DNA mismatch repair (MMR). MMR starts when a MutS homolog recognizes a mismatch and undergoes an ATP-dependent transformation to an elusive sliding clamp state. How this transient state promotes MutL homolog recruitment and activation of repair is unclear. Here we present a crystal structure of the MutS/MutL complex using a site-specifically crosslinked complex and examine how large conformational changes lead to activation of MutL. The structure captures MutS in the sliding clamp conformation, where tilting of the MutS subunits across each other pushes DNA into a new channel, and reorientation of the connector domain creates an interface for MutL with both MutS subunits. Our work explains how the sliding clamp promotes loading of MutL onto DNA, to activate downstream effectors. We thus elucidate a crucial mechanism that ensures that MMR is initiated only after detection of a DNA mismatch.
Asunto(s)
Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , ADN/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/química , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/metabolismo , Cristalografía por Rayos X , Modelos Moleculares , Proteínas MutL , Unión Proteica , Conformación ProteicaRESUMEN
Homeologous recombination between divergent DNA sequences is inhibited by DNA mismatch repair. In Escherichia coli, MutS and MutL respond to DNA mismatches within recombination intermediates and prevent strand exchange via an unknown mechanism. Here, using purified proteins and DNA substrates, we find that in addition to mismatches within the heteroduplex region, secondary structures within the displaced single-stranded DNA formed during branch migration within the recombination intermediate are involved in the inhibition. We present a model that explains how higher-order complex formation of MutS, MutL, and DNA blocks branch migration by preventing rotation of the DNA strands within the recombination intermediate. Furthermore, we find that the helicase UvrD is recruited to directionally resolve these trapped intermediates toward DNA substrates. Thus, our results explain on a mechanistic level how the coordinated action between MutS, MutL, and UvrD prevents homeologous recombination and maintains genome stability.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , ADN Helicasas/metabolismo , Reparación de la Incompatibilidad de ADN , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Proteínas de Escherichia coli/metabolismo , Recombinación Homóloga/genética , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/metabolismo , ADN de Cadena Simple/metabolismo , Escherichia coli/genética , Variación Genética , Proteínas MutL , Rec A Recombinasas/metabolismoRESUMEN
The process of DNA mismatch repair is initiated when MutS recognizes mismatched DNA bases and starts the repair cascade. The Escherichia coli MutS protein exists in an equilibrium between dimers and tetramers, which has compromised biophysical analysis. To uncouple these states, we have generated stable dimers and tetramers, respectively. These proteins allowed kinetic analysis of DNA recognition and structural analysis of the full-length protein by X-ray crystallography and small angle X-ray scattering. Our structural data reveal that the tetramerization domains are flexible with respect to the body of the protein, resulting in mostly extended structures. Tetrameric MutS has a slow dissociation from DNA, which can be due to occasional bending over and binding DNA in its two binding sites. In contrast, the dimer dissociation is faster, primarily dependent on a combination of the type of mismatch and the flanking sequence. In the presence of ATP, we could distinguish two kinetic groups: DNA sequences where MutS forms sliding clamps and those where sliding clamps are not formed efficiently. Interestingly, this inability to undergo a conformational change rather than mismatch affinity is correlated with mismatch repair.
Asunto(s)
Disparidad de Par Base , ADN/química , Proteínas de Escherichia coli/química , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/química , Adenosina Trifosfato/metabolismo , ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/metabolismo , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de ProteínaRESUMEN
The DNA mismatch repair protein MutS recognizes mispaired bases in DNA and initiates repair in an ATP-dependent manner. Understanding of the allosteric coupling between DNA mismatch recognition and two asymmetric nucleotide binding sites at opposing sides of the MutS dimer requires identification of the relevant MutS.mmDNA.nucleotide species. Here, we use native mass spectrometry to detect simultaneous DNA mismatch binding and asymmetric nucleotide binding to Escherichia coli MutS. To resolve the small differences between macromolecular species bound to different nucleotides, we developed a likelihood based algorithm capable to deconvolute the observed spectra into individual peaks. The obtained mass resolution resolves simultaneous binding of ADP and AMP.PNP to this ABC ATPase in the absence of DNA. Mismatched DNA regulates the asymmetry in the ATPase sites; we observe a stable DNA-bound state containing a single AMP.PNP cofactor. This is the first direct evidence for such a postulated mismatch repair intermediate, and showcases the potential of native MS analysis in detecting mechanistically relevant reaction intermediates.
Asunto(s)
Disparidad de Par Base , ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/metabolismo , Adenilil Imidodifosfato/metabolismo , Algoritmos , Sitios de Unión , ADN/química , Dimerización , Nucleótidos/metabolismo , Unión Proteica , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
The DNA mismatch repair protein MutS acts as a molecular switch. It toggles between ADP and ATP states and is regulated by mismatched DNA. This is analogous to G-protein switches and the regulation of their "on" and "off" states by guanine exchange factors. Although GDP release in monomeric GTPases is accelerated by guanine exchange factor-induced removal of magnesium from the catalytic site, we found that release of ADP from MutS is not influenced by the metal ion in this manner. Rather, ADP release is induced by the binding of mismatched DNA at the opposite end of the protein, a long-range allosteric response resembling the mechanism of activation of heterotrimeric GTPases. Magnesium influences switching in MutS by inducing faster and tighter ATP binding, allowing rapid downstream responses. MutS mutants with decreased affinity for the metal ion are impaired in fast switching and in vivo mismatch repair. Thus, the G-proteins and MutS conceptually employ the same efficient use of the high energy cofactor: slow hydrolysis in the absence of a signal and fast conversion to the active state when required.
Asunto(s)
Adenosina Difosfato/química , Adenosina Trifosfato/química , ADN Bacteriano/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Magnesio/química , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/química , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Regulación Alostérica , Dominio Catalítico/fisiología , Reparación de la Incompatibilidad de ADN/fisiología , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Hidrólisis , Magnesio/metabolismo , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/genética , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/metabolismoRESUMEN
MutS plays a critical role in DNA mismatch repair in Escherichia coli by binding to mismatches and initiating repair in an ATP-dependent manner. Mutational analysis of a highly conserved glutamate, Glu38, has revealed its role in mismatch recognition by enabling MutS to discriminate between homoduplex and mismatched DNA. Crystal structures of MutS have shown that Glu38 forms a hydrogen bond to one of the mismatched bases. In this study, we have analyzed the crystal structures, DNA binding and the response to ATP binding of three Glu38 mutants. While confirming the role of the negative charge in initial discrimination, we show that in vivo mismatch repair can proceed even when discrimination is low. We demonstrate that the formation of a hydrogen bond by residue 38 to the mismatched base authorizes repair by inducing intramolecular signaling, which results in the inhibition of rapid hydrolysis of distally bound ATP. This allows formation of the stable MutS-ATP-DNA clamp, a key intermediate in triggering downstream repair events.
Asunto(s)
Disparidad de Par Base/genética , Reparación del ADN/fisiología , Ácido Glutámico/metabolismo , Modelos Moleculares , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/química , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/metabolismo , Adenosina Trifosfato/metabolismo , Disparidad de Par Base/fisiología , Calorimetría , Cristalografía , Ensayo de Cambio de Movilidad Electroforética , Escherichia coli , Ácido Glutámico/química , Enlace de Hidrógeno , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/genética , Mutagénesis Sitio-Dirigida , Oligonucleótidos , Resonancia por Plasmón de SuperficieRESUMEN
MutS is the key protein of the Escherichia coli DNA mismatch repair system. It recognizes mispaired and unpaired bases and has intrinsic ATPase activity. ATP binding after mismatch recognition by MutS serves as a switch that enables MutL binding and the subsequent initiation of mismatch repair. However, the mechanism of this switch is poorly understood. We have investigated the effects of ATP binding on the MutS structure. Crystallographic studies of ATP-soaked crystals of MutS show a trapped intermediate, with ATP in the nucleotide-binding site. Local rearrangements of several residues around the nucleotide-binding site suggest a movement of the two ATPase domains of the MutS dimer toward each other. Analytical ultracentrifugation experiments confirm such a rearrangement, showing increased affinity between the ATPase domains upon ATP binding and decreased affinity in the presence of ADP. Mutations of specific residues in the nucleotide-binding domain reduce the dimer affinity of the ATPase domains. In addition, ATP-induced release of DNA is strongly reduced in these mutants, suggesting that the two activities are coupled. Hence, it seems plausible that modulation of the affinity between ATPase domains is the driving force for conformational changes in the MutS dimer. These changes are driven by distinct amino acids in the nucleotide-binding site and form the basis for long-range interactions between the ATPase domains and DNA-binding domains and subsequent binding of MutL and initiation of mismatch repair.
Asunto(s)
Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Disparidad de Par Base/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Sustitución de Aminoácidos , Cristalografía por Rayos X , Proteínas de Escherichia coli , Modelos Moleculares , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN , Mutagénesis Sitio-Dirigida , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
We have refined a series of isomorphous crystal structures of the Escherichia coli DNA mismatch repair enzyme MutS in complex with G:T, A:A, C:A and G:G mismatches and also with a single unpaired thymidine. In all these structures, the DNA is kinked by approximately 60 degrees upon protein binding. Two residues widely conserved in the MutS family are involved in mismatch recognition. The phenylalanine, Phe 36, is seen stacking on one of the mismatched bases. The same base is also seen forming a hydrogen bond to the glutamate Glu 38. This hydrogen bond involves the N7 if the base stacking on Phe 36 is a purine and the N3 if it is a pyrimidine (thymine). Thus, MutS uses a common binding mode to recognize a wide range of mismatches.
Asunto(s)
Adenosina Trifosfatasas/química , Proteínas Bacterianas/química , Disparidad de Par Base , Proteínas de Unión al ADN/química , ADN/química , Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/metabolismo , Sitios de Unión , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Escherichia coli/enzimología , Proteínas de Escherichia coli , Ácido Glutámico/química , Ácido Glutámico/metabolismo , Enlace de Hidrógeno , Modelos Moleculares , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN , Fenilalanina/química , Fenilalanina/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Especificidad por SustratoRESUMEN
DNA mismatch repair is an essential safeguard of genomic integrity by removing base mispairings that may arise from DNA polymerase errors or from homologous recombination between DNA strands. In Escherichia coli, the MutS enzyme recognizes mismatches and initiates repair. MutS has an intrinsic ATPase activity crucial for its function, but which is poorly understood. We show here that within the MutS homodimer, the two chemically identical ATPase sites have different affinities for ADP, and the two sites alternate in ATP hydrolysis. A single residue, Arg697, located at the interface of the two ATPase domains, controls the asymmetry. When mutated, the asymmetry is lost and mismatch repair in vivo is impaired. We propose that asymmetry of the ATPase domains is an essential feature of mismatch repair that controls the timing of the different steps in the repair cascade.