The article investigates the mechanism of DNA replication initiation in eukaryotes using cryo-EM. It focuses on the role of Mcm10 in splitting the double CMGE (CMG-MCM complex) into two single CMGEs, which is crucial for DNA unwinding and replication fork progression. The study reveals that Mcm10 binds to the N-terminal face of MCM, engaging the homo-dimerization interface and splitting the dCMGE. This process prevents re-dimerization of the MCM hexamers, allowing the helicases to move toward each other and cross paths. The C-terminal domain of Mcm10 also supports this process by increasing the affinity of the interaction with the CMGE. The N-terminal Mcm10 is responsible for initiating DNA unwinding from the N-terminal side of MCM, while the C-terminal Mcm10 is involved in stimulating fork rate and modulating replication fork progression. The study provides insights into the structural changes that occur during the transition from dCMGE to sCMGE, including the narrowing of the MCM channel and the establishment of new contacts with the leading-strand template. These findings contribute to a better understanding of the molecular mechanisms underlying eukaryotic DNA replication initiation and elongation.The article investigates the mechanism of DNA replication initiation in eukaryotes using cryo-EM. It focuses on the role of Mcm10 in splitting the double CMGE (CMG-MCM complex) into two single CMGEs, which is crucial for DNA unwinding and replication fork progression. The study reveals that Mcm10 binds to the N-terminal face of MCM, engaging the homo-dimerization interface and splitting the dCMGE. This process prevents re-dimerization of the MCM hexamers, allowing the helicases to move toward each other and cross paths. The C-terminal domain of Mcm10 also supports this process by increasing the affinity of the interaction with the CMGE. The N-terminal Mcm10 is responsible for initiating DNA unwinding from the N-terminal side of MCM, while the C-terminal Mcm10 is involved in stimulating fork rate and modulating replication fork progression. The study provides insights into the structural changes that occur during the transition from dCMGE to sCMGE, including the narrowing of the MCM channel and the establishment of new contacts with the leading-strand template. These findings contribute to a better understanding of the molecular mechanisms underlying eukaryotic DNA replication initiation and elongation.