RESUMO
The regulated loading of the Mcm2-7 DNA helicase (comprising six related subunits, Mcm2 to Mcm7) into pre-replicative complexes at multiple replication origins ensures precise once per cell cycle replication in eukaryotic cells. The origin recognition complex (ORC), Cdc6 and Cdt1 load Mcm2-7 into a double hexamer bound around duplex DNA in an ATP-dependent reaction, but the molecular mechanism of this origin 'licensing' is still poorly understood. Here we show that both Mcm2-7 hexamers in Saccharomyces cerevisiae are recruited to origins by an essential, conserved carboxy-terminal domain of Mcm3 that interacts with and stimulates the ATPase activity of ORC-Cdc6. ATP hydrolysis can promote Mcm2-7 loading, but can also promote Mcm2-7 release if components are missing or if ORC has been inactivated by cyclin-dependent kinase phosphorylation. Our work provides new insights into how origins are licensed and reveals a novel ATPase-dependent mechanism contributing to precise once per cell cycle replication.
Assuntos
Adenosina Trifosfatases/metabolismo , Replicação do DNA/genética , Origem de Replicação/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Hidrólise , Componente 3 do Complexo de Manutenção de Minicromossomo , Componente 7 do Complexo de Manutenção de Minicromossomo , Proteínas Nucleares/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de SequênciaRESUMO
Licensing of origins of eukaryotic DNA replication involves the loading of six minichromosome maintenance proteins (Mcm2-7) into pre-replicative complexes (pre-RCs). The assembly of the pre-RC is restricted to G1 phase of the cell cycle, which is crucial to ensure once per cell cycle DNA replication. Mcm2-7 is loaded by the action of the origin recognition complex (ORC), Cdc6 and Cdt1 and requires ATP. In vitro reconstitution of this reaction has shown that Mcm2-7 is loaded onto DNA as a symmetrical head-to-head double hexamer. We describe in detail how pre-RC proteins are purified and used to reconstitute pre-RC formation in vitro. This method is useful for studying the biochemical mechanisms of Mcm2-7 loading as well as subsequent steps in DNA replication.