Abstract: The Cdc45-Mcm2-7-GINS (CMG) complex is the replication fork helicase in eukaryotes. Synthetic lethal with Dpb11-1 (Sld2) is required for the initiation of DNA replication, and the S phase cyclin-dependent kinase (S-CDK) phosphorylates Sld2 in vivo. We purified components of the replication initiation machinery and studied their interactions in vitro. We found that unphosphorylated or CDK-phosphorylated Sld2 binds to the mini chromosome maintenance (Mcm)2-7 complex with similar efficiency. Sld2 interaction with Mcm2-7 blocks the interaction between GINS and Mcm2-7. The interaction between CDK-phosphorylated Sld2 and Mcm2-7 is substantially inhibited by origin single-stranded DNA (ssDNA). Furthermore, origin ssDNA allows GINS to bind to Mcm2-7 in the presence of CDK-phosphorylated Sld2. However, unphosphorylated Sld2 blocks the interaction between GINS and Mcm2-7 even in the presence of origin ssDNA. We identified a mutant of Sld2 that does not bind to DNA. When this mutant is expressed in yeast cells, cell growth is severely inhibited with very slow progression into S phase. We propose a model wherein Sld2 blocks the interaction between GINS and Mcm2-7 in vivo. Once origin ssDNA is extruded from the Mcm2-7 ring and CDK phosphorylates Sld2, the origin ssDNA binds to CDK-phosphorylated Sld2. This event may allow the interaction between GINS and Mcm2-7 in vivo. Thus, CDK phosphorylation of Sld2 may be important to release Sld2 from Mcm2-7, thereby allowing GINS to bind Mcm2-7. Furthermore, origin ssDNA may stimulate the formation of the CMG complex by alleviating inhibitory interactions between Sld2 with Mcm2-7.