RESUMEN
DNA replication in eukaryotic cells initiates from replication origins that bind the Origin Recognition Complex (ORC). Origin establishment requires well-defined DNA sequence motifs in Saccharomyces cerevisiae and some other budding yeasts, but most eukaryotes lack sequence-specific origins. A 3.9 Å structure of S. cerevisiae ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) bound to origin DNA revealed that a loop within Orc2 inserts into a DNA minor groove and an α-helix within Orc4 inserts into a DNA major groove. Using a massively parallel origin selection assay coupled with a custom mutual-information-based modeling approach, and a separate analysis of whole-genome replication profiling, here we show that the Orc4 α-helix contributes to the DNA sequence-specificity of origins in S. cerevisiae and Orc4 α-helix mutations change genome-wide origin firing patterns. The DNA sequence specificity of replication origins, mediated by the Orc4 α-helix, has co-evolved with the gain of ORC-Sir4-mediated gene silencing and the loss of RNA interference.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Silenciador del Gen , Complejo de Reconocimiento del Origen/metabolismo , Origen de Réplica/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Replicación del ADN , ADN de Hongos/genética , Evolución Molecular , Mutación , Complejo de Reconocimiento del Origen/ultraestructura , Conformación Proteica en Hélice alfa/genética , Proteínas de Saccharomyces cerevisiae/ultraestructura , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Especificidad por Sustrato/genéticaRESUMEN
In preparation for bidirectional DNA replication, the origin recognition complex (ORC) loads two hexameric MCM helicases to form a head-to-head double hexamer around DNA1,2. The mechanism of MCM double-hexamer formation is debated. Single-molecule experiments have suggested a sequential mechanism, in which the ORC-dependent loading of the first hexamer drives the recruitment of the second hexamer3. By contrast, biochemical data have shown that two rings are loaded independently via the same ORC-mediated mechanism, at two inverted DNA sites4,5. Here we visualize MCM loading using time-resolved electron microscopy, and identify intermediates in the formation of the double hexamer. We confirm that both hexamers are recruited via the same interaction that occurs between ORC and the C-terminal domains of the MCM helicases. Moreover, we identify the mechanism of coupled MCM loading. The loading of the first MCM hexamer around DNA creates a distinct interaction site, which promotes the engagement of ORC at the N-terminal homodimerization interface of MCM. In this configuration, ORC is poised to direct the recruitment of the second hexamer in an inverted orientation, which is suitable for the formation of the double hexamer. Our results therefore reconcile the two apparently contrasting models derived from single-molecule experiments and biochemical data.
Asunto(s)
Microscopía por Crioelectrón , Modelos Moleculares , Complejo de Reconocimiento del Origen/metabolismo , Complejo de Reconocimiento del Origen/ultraestructura , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestructura , Simulación por Computador , Nucleosomas/metabolismo , Nucleosomas/ultraestructura , Complejo de Reconocimiento del Origen/química , Unión Proteica , Estructura Cuaternaria de Proteína , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
The six-subunit origin recognition complex (ORC) binds to DNA to mark the site for the initiation of replication in eukaryotes. Here we report a 3 Å cryo-electron microscopy structure of the Saccharomyces cerevisiae ORC bound to a 72-base-pair origin DNA sequence that contains the ARS consensus sequence (ACS) and the B1 element. The ORC encircles DNA through extensive interactions with both phosphate backbone and bases, and bends DNA at the ACS and B1 sites. Specific recognition of thymine residues in the ACS is carried out by a conserved basic amino acid motif of Orc1 in the minor groove, and by a species-specific helical insertion motif of Orc4 in the major groove. Moreover, similar insertions into major and minor grooves are also embedded in the B1 site by basic patch motifs from Orc2 and Orc5, respectively, to contact bases and to bend DNA. This work pinpoints a conserved role of ORC in modulating DNA structure to facilitate origin selection and helicase loading in eukaryotes.
Asunto(s)
Microscopía por Crioelectrón , Complejo de Reconocimiento del Origen/química , Complejo de Reconocimiento del Origen/ultraestructura , Origen de Réplica , Saccharomyces cerevisiae , Secuencia de Bases , ADN/química , ADN/genética , ADN/metabolismo , ADN/ultraestructura , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Modelos Moleculares , Complejo de Reconocimiento del Origen/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/ultraestructura , Especificidad por SustratoRESUMEN
A family of six homologous subunits, Mcm2, -3, -4, -5, -6, and -7, each with its own unique features, forms the catalytic core of the eukaryotic replicative helicase. The necessity of six similar but non-identical subunits has been a mystery since its initial discovery. Recent cryo-EM structures of the Mcm2-7 (MCM) double hexamer, its precursors, and the origin recognition complex (ORC)-Cdc6-Cdt1-Mcm2-7 (OCCM) intermediate showed that each of these subunits plays a distinct role in orchestrating the assembly of the pre-replication complex (pre-RC) by ORC-Cdc6 and Cdt1.
Asunto(s)
Replicación del ADN , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Complejo de Reconocimiento del Origen/metabolismo , Animales , Dominio Catalítico , Proteínas de Ciclo Celular/metabolismo , Humanos , Proteínas de Mantenimiento de Minicromosoma/química , Proteínas de Mantenimiento de Minicromosoma/ultraestructura , Modelos Moleculares , Complejos Multiproteicos , Proteínas Nucleares/metabolismo , Conformación de Ácido Nucleico , Complejo de Reconocimiento del Origen/química , Complejo de Reconocimiento del Origen/ultraestructura , Unión Proteica , Subunidades de Proteína , Relación Estructura-ActividadRESUMEN
In eukaryotes, DNA replication requires the origin recognition complex (ORC), a six-subunit assembly that promotes replisome formation on chromosomal origins. Despite extant homology between certain subunits, the degree of structural and organizational overlap between budding yeast and metazoan ORC has been unclear. Using 3D electron microscopy, we determined the subunit organization of metazoan ORC, revealing that it adopts a global architecture very similar to the budding yeast complex. Bioinformatic analysis extends this conservation to Orc6, a subunit of somewhat enigmatic function. Unexpectedly, a mutation in the Orc6 C-terminus linked to Meier-Gorlin syndrome, a dwarfism disorder, impedes proper recruitment of Orc6 into ORC; biochemical studies reveal that this region of Orc6 associates with a previously uncharacterized domain of Orc3 and is required for ORC function and MCM2-7 loading in vivo. Together, our results suggest that Meier-Gorlin syndrome mutations in Orc6 impair the formation of ORC hexamers, interfering with appropriate ORC functions. DOI:http://dx.doi.org/10.7554/eLife.00882.001.
Asunto(s)
Microtia Congénita/genética , Trastornos del Crecimiento/genética , Micrognatismo/genética , Mutación , Complejo de Reconocimiento del Origen/genética , Rótula/anomalías , Animales , Drosophila , Humanos , Microscopía Electrónica , Complejo de Reconocimiento del Origen/ultraestructuraRESUMEN
Binding of Cdc6 to the origin recognition complex (ORC) is a key step in the assembly of a pre-replication complex (pre-RC) at origins of DNA replication. ORC recognizes specific origin DNA sequences in an ATP-dependent manner. Here we demonstrate cooperative binding of Saccharomyces cerevisiae Cdc6 to ORC on DNA in an ATP-dependent manner, which induces a change in the pattern of origin binding that requires the Orc1 ATPase. The reaction is blocked by specific origin mutations that do not interfere with the interaction between ORC and DNA. Single-particle reconstruction of electron microscopic images shows that the ORC-Cdc6 complex forms a ring-shaped structure with dimensions similar to those of the ring-shaped MCM helicase. The ORC-Cdc6 structure is predicted to contain six AAA+ subunits, analogous to other ATP-dependent protein machines. We suggest that Cdc6 and origin DNA activate a molecular switch in ORC that contributes to pre-RC assembly.