biostudies-literatureLi HJapan Agency for Medical Research and Development, Platform Project for Supporting Drug Discovery and Life Science ResearchJapan Society for the Promotion of Science, KAKENHI Grant-in-aid for Scientific Research(B)RP101717https://www.ebi.ac.uk/biostudies/studies/S-EPMC12416888biostudies-literatureUnknown13DNA replication requires recruitment of Cdc45 and GINS into the MCM double hexamer by initiation factors to form an active helicase, the Cdc45-MCM-GINS (CMG) complex, at the replication origins. The initiation factor Sld3 is a central regulator of Cdc45 and GINS recruitment, working with Sld7 together. However, the mechanism through which Sld3 regulates CMG complex formation remains unclear. Here, we present the structure of the Sld3 Cdc45-binding domain in complex with Cdc45 (Sld3CBD-Cdc45), showing detailed interactions and conformational changes required for binding to each other. The mutant analysis indicated that the binding between Sld3CBD and Cdc45 could be broken easily. We also revealed that Sld3CBD, GINS, and MCM bind to different sites on Cdc45 in the Sld3CBD-CMG model, indicatieLifeSld3CBD-Cdc45 structural insights into Cdc45 recruitment for CMG complex formation during DNA replication.PMC12416888JP18am010107121H01754JP19am0101083Araki HLi HItou HKato KMuramatsu SIshizaki IYao MOse TSun XfalseSld3CBD-Cdc45 structural insights into Cdc45 recruitment for CMG complex formation during DNA replication.DNA replication requires recruitment of Cdc45 and GINS into the MCM double hexamer by initiation factors to form an active helicase, the Cdc45-MCM-GINS (CMG) complex, at the replication origins. The initiation factor Sld3 is a central regulator of Cdc45 and GINS recruitment, working with Sld7 together. However, the mechanism through which Sld3 regulates CMG complex formation remains unclear. Here, we present the structure of the Sld3 Cdc45-binding domain in complex with Cdc45 (Sld3CBD-Cdc45), showing detailed interactions and conformational changes required for binding to each other. The mutant analysis indicated that the binding between Sld3CBD and Cdc45 could be broken easily. We also revealed that Sld3CBD, GINS, and MCM bind to different sites on Cdc45 in the Sld3CBD-CMG model, indicati2025-01-01T00:00:00Z2025 Sep2026-06-02T02:29:49.725Z2026-04-13T03:12:35.328ZS-EPMC124168884092007510.7554/eLife.101717