A unique DNA entry gate serves for regulated loading of the eukaryotic replicative helicase MCM2-7 onto DNA

• Published in: Genes & development Vol. 28; no. 15; pp. 1653 - 1666
• Main Authors: Samel, Stefan A, Fernández-Cid, Alejandra, Sun, Jingchuan, Riera, Alberto, Tognetti, Silvia, Herrera, M Carmen, Li, Huilin, Speck, Christian
• Format: Journal Article
• Language: English
• Published: United States Cold Spring Harbor Laboratory Press 01.08.2014
• Subjects: Adenosine Triphosphate - metabolism; Cell Cycle; Chromosomes, Fungal - metabolism; DNA, Fungal - metabolism; Enzyme Activation; Hydrolysis; Minichromosome Maintenance Proteins - chemistry; Minichromosome Maintenance Proteins - genetics; Minichromosome Maintenance Proteins - metabolism; Minichromosome Maintenance Proteins - ultrastructure; Multiprotein Complexes - chemistry; Multiprotein Complexes - genetics; Multiprotein Complexes - ultrastructure; Protein Subunits - chemistry; Protein Subunits - genetics; Protein Subunits - metabolism; Replication Origin - physiology; Research Paper; Saccharomyces cerevisiae; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; pre-RC; DNA replication; replicative helicase; cancer; DNA licensing; genomic stability
• ISSN: 0890-9369; 1549-5477
• DOI: 10.1101/gad.242404.114

The regulated loading of the replicative helicase minichromosome maintenance proteins 2-7 (MCM2-7) onto replication origins is a prerequisite for replication fork establishment and genomic stability. Origin recognition complex (ORC), Cdc6, and Cdt1 assemble two MCM2-7 hexamers into one double hexamer around dsDNA. Although the MCM2-7 hexamer can adopt a ring shape with a gap between Mcm2 and Mcm5, it is unknown which Mcm interface functions as the DNA entry gate during regulated helicase loading. Here, we establish that the Saccharomyces cerevisiae MCM2-7 hexamer assumes a closed ring structure, suggesting that helicase loading requires active ring opening. Using a chemical biology approach, we show that ORC-Cdc6-Cdt1-dependent helicase loading occurs through a unique DNA entry gate comprised of the Mcm2 and Mcm5 subunits. Controlled inhibition of DNA insertion triggers ATPase-driven complex disassembly in vitro, while in vivo analysis establishes that Mcm2/Mcm5 gate opening is essential for both helicase loading onto chromatin and cell cycle progression. Importantly, we demonstrate that the MCM2-7 helicase becomes loaded onto DNA as a single hexamer during ORC/Cdc6/Cdt1/MCM2-7 complex formation prior to MCM2-7 double hexamer formation. Our study establishes the existence of a unique DNA entry gate for regulated helicase loading, revealing key mechanisms in helicase loading, which has important implications for helicase activation.