Sld3CBD-Cdc45 structural insights into Cdc45 recruitment for CMG complex formation on DNA replication

Abstract

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, indicating that after recruitment of Cdc45, Sld7-Sld3 could remain in Cdc45-MCM until CMG formation. The consistency between the particle size of Sld7-Sld3-Cdc45 and the distance between Sld3CBDs in the Cdc45-MCM dimer indicated the binding manner of the Cdc45-Sld3-[Sld7]2-Sld3-Cdc45 off/on MCM double hexamer. A DNA-binding assay of Sld3 and its complexes with single-stranded ARS1 fragments revealed a relationship between the dissociation of Sld7-Sld3 from CMG and the unwound single-stranded DNA. These findings help to further our understanding of the molecular basis of the regulation of CMG complex formation by Sld3.