A study using cryo-EM reveals the molecular mechanism of eukaryotic DNA replication initiation. The minichromosome maintenance (MCM) helicase, a hexameric ring, is initially loaded onto duplex DNA at origins of replication as a double hexamer with dimerizing N-terminal domains. Upon S-phase transition, the double hexamer is converted into a double CMGE (Cdc45-MCM-GINS-Pole) structure, which partially separates the two MCM hexamers and nucleates DNA melting. Mcm10, a single-stranded DNA-binding protein, recruits to the double CMGE and splits it into two single CMGEs (sCMGEs), allowing the helicases to move apart and expose duplex DNA between them. Mcm10 binds to the N-terminal homo-dimerization interface of MCM, facilitating the separation of the double CMGE and promoting helicase crossing. The sCMGEs then transition from duplex to single-stranded DNA binding, enabling DNA unwinding. Mcm10's C-terminal domain also interacts with the MCM ATPase, supporting the splitting of the double CMGE and the initiation of DNA replication. The study shows that Mcm10's N-terminal domain is essential for splitting the double CMGE, while its C-terminal domain is involved in fork rate stimulation. The results indicate that Mcm10 plays a critical structural role in the initiation of DNA replication by splitting the double CMGE and promoting helicase crossing. The study also highlights the importance of Mcm10 in ensuring that DNA is replicated only once per cell cycle, preventing chromosome instability and cancer. The findings provide insights into the molecular mechanisms underlying DNA replication initiation in eukaryotic cells.A study using cryo-EM reveals the molecular mechanism of eukaryotic DNA replication initiation. The minichromosome maintenance (MCM) helicase, a hexameric ring, is initially loaded onto duplex DNA at origins of replication as a double hexamer with dimerizing N-terminal domains. Upon S-phase transition, the double hexamer is converted into a double CMGE (Cdc45-MCM-GINS-Pole) structure, which partially separates the two MCM hexamers and nucleates DNA melting. Mcm10, a single-stranded DNA-binding protein, recruits to the double CMGE and splits it into two single CMGEs (sCMGEs), allowing the helicases to move apart and expose duplex DNA between them. Mcm10 binds to the N-terminal homo-dimerization interface of MCM, facilitating the separation of the double CMGE and promoting helicase crossing. The sCMGEs then transition from duplex to single-stranded DNA binding, enabling DNA unwinding. Mcm10's C-terminal domain also interacts with the MCM ATPase, supporting the splitting of the double CMGE and the initiation of DNA replication. The study shows that Mcm10's N-terminal domain is essential for splitting the double CMGE, while its C-terminal domain is involved in fork rate stimulation. The results indicate that Mcm10 plays a critical structural role in the initiation of DNA replication by splitting the double CMGE and promoting helicase crossing. The study also highlights the importance of Mcm10 in ensuring that DNA is replicated only once per cell cycle, preventing chromosome instability and cancer. The findings provide insights into the molecular mechanisms underlying DNA replication initiation in eukaryotic cells.