RESUMEN
The Origin Recognition Complex (ORC) binds to sites in chromosomes to specify the location of origins of DNA replication. The S. cerevisiae ORC binds to specific DNA sequences throughout the cell cycle but becomes active only when it binds to the replication initiator Cdc6. It has been unclear at the molecular level how Cdc6 activates ORC, converting it to an active recruiter of the Mcm2-7 hexamer, the core of the replicative helicase. Here we report the cryo-EM structure at 3.3 Å resolution of the yeast ORC-Cdc6 bound to an 85-bp ARS1 origin DNA. The structure reveals that Cdc6 contributes to origin DNA recognition via its winged helix domain (WHD) and its initiator-specific motif. Cdc6 binding rearranges a short α-helix in the Orc1 AAA+ domain and the Orc2 WHD, leading to the activation of the Cdc6 ATPase and the formation of the three sites for the recruitment of Mcm2-7, none of which are present in ORC alone. The results illuminate the molecular mechanism of a critical biochemical step in the licensing of eukaryotic replication origins.
Asunto(s)
Proteínas de Ciclo Celular/genética , Replicación del ADN/genética , ADN de Hongos/genética , Complejo de Reconocimiento del Origen/genética , Origen de Réplica/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Microscopía por Crioelectrón , ADN de Hongos/química , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Modelos Moleculares , Conformación de Ácido Nucleico , Complejo de Reconocimiento del Origen/química , Complejo de Reconocimiento del Origen/metabolismo , Unión Proteica , Dominios Proteicos , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
We present a concise workflow to enhance the mass spectrometric detection of crosslinked peptides by introducing sequential digestion and the crosslink identification software xiSEARCH. Sequential digestion enhances peptide detection by selective shortening of long tryptic peptides. We demonstrate our simple 12-fraction protocol for crosslinked multi-protein complexes and cell lysates, quantitative analysis, and high-density crosslinking, without requiring specific crosslinker features. This overall approach reveals dynamic protein-protein interaction sites, which are accessible, have fundamental functional relevance and are therefore ideally suited for the development of small molecule inhibitors.
Asunto(s)
Espectrometría de Masas/métodos , Mapeo de Interacción de Proteínas/métodos , Proteínas/química , Proteómica/métodos , Citosol/química , Humanos , Células K562 , Modelos Moleculares , Fragmentos de Péptidos/química , Conformación Proteica , Programas InformáticosRESUMEN
DNA replication results in the doubling of the genome prior to cell division. This process requires the assembly of 50 or more protein factors into a replication fork. Here, we review recent structural and biochemical insights that start to explain how specific proteins recognize DNA replication origins, load the replicative helicase on DNA, unwind DNA, synthesize new DNA strands, and reassemble chromatin. We focus on the minichromosome maintenance (MCM2-7) proteins, which form the core of the eukaryotic replication fork, as this complex undergoes major structural rearrangements in order to engage with DNA, regulate its DNA-unwinding activity, and maintain genome stability.
Asunto(s)
Replicación del ADN/fisiología , Animales , Cromatina/metabolismo , ADN Helicasas/metabolismo , Replicación del ADN/genética , Evolución Molecular , Inestabilidad Genómica/genética , Humanos , Proteínas de Mantenimiento de Minicromosoma/genética , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Origen de Réplica/fisiologíaRESUMEN
To initiate DNA replication, the origin recognition complex (ORC) and Cdc6 load an Mcm2-7 double hexamer onto DNA. Without ATP hydrolysis, ORC-Cdc6 recruits one Cdt1-bound Mcm2-7 hexamer, thus forming an ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) helicase-loading intermediate. Here we report a 3.9-Å structure of Saccharomyces cerevisiae OCCM on DNA. Flexible Mcm2-7 winged-helix domains (WHDs) engage ORC-Cdc6. A three-domain Cdt1 configuration embraces Mcm2, Mcm4, and Mcm6, thus comprising nearly half of the hexamer. The Cdt1 C-terminal domain extends to the Mcm6 WHD, which binds the Orc4 WHD. DNA passes through the ORC-Cdc6 and Mcm2-7 rings. Origin DNA interaction is mediated by an α-helix within Orc4 and positively charged loops within Orc2 and Cdc6. The Mcm2-7 C-tier AAA+ ring is topologically closed by an Mcm5 loop that embraces Mcm2, but the N-tier-ring Mcm2-Mcm5 interface remains open. This structure suggests a loading mechanism of the first Cdt1-bound Mcm2-7 hexamer by ORC-Cdc6.
Asunto(s)
Proteínas de Ciclo Celular/química , Replicación del ADN , Proteínas de Unión al ADN/química , Proteínas de Mantenimiento de Minicromosoma/química , Origen de Réplica , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Sitios de Unión , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/ultraestructura , Microscopía por Crioelectrón , ADN de Hongos/química , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/ultraestructura , Espectrometría de Masas , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Proteínas de Mantenimiento de Minicromosoma/ultraestructura , Modelos Moleculares , Nucleótidos/metabolismo , Unión Proteica , Dominios Proteicos , Multimerización de Proteína , Estructura Secundaria de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestructuraRESUMEN
Mixed Lineage Leukemia 5 (MLL5) plays a key role in hematopoiesis, spermatogenesis and cell cycle progression. Chromatin binding is ensured by its plant homeodomain (PHD) through a direct interaction with the N-terminus of histone H3 (H3). In addition, MLL5 contains a Su(var)3-9, Enhancer of zeste, Trithorax (SET) domain, a protein module that usually displays histone lysine methyltransferase activity. We report here the crystal structure of the unliganded SET domain of human MLL5 at 2.1 Å resolution. Although it shows most of the canonical features of other SET domains, both the lack of key residues and the presence in the SET-I subdomain of an unusually large loop preclude the interaction of MLL5 SET with its cofactor and substrate. Accordingly, we show that MLL5 is devoid of any in vitro methyltransferase activity on full-length histones and histone H3 peptides. Hence, the three dimensional structure of MLL5 SET domain unveils the structural basis for its lack of methyltransferase activity and suggests a new regulatory mechanism.
