title: GINS and Sld3 compete with one another for Mcm2-7 and Cdc45 binding.
abstract: Sld3 is essential for the initiation of DNA replication, but Sld3 does not travel with a replication fork. GINS binds to Cdc45 and Mcm2-7 to form the replication fork helicase in eukaryotes. We purified Sld3, Cdc45, GINS, and Mcm2-7 and studied their interaction and assembly into complexes. Sld3 binds tightly to Cdc45 in the presence or absence of cyclin-dependent kinase activity. Furthermore, Sld3 binds tightly to the Mcm2-7 complex, and a ternary complex forms among Cdc45, Mcm2-7, and Sld3, with a 1:1:1 stoichiometry (CMS complex). GINS binds directly to Mcm2-7, and GINS competes with Sld3 for Mcm2-7 binding. GINS also binds directly to Cdc45, and GINS competes with Sld3 for Cdc45 binding. Cdc45, Mcm2-7, and GINS form a ternary complex with a stoichiometry of 1:1:1 (CMG complex). Size exclusion data reveal that when Sld3, Cdc45, Mcm2-7, and GINS are added together, the result is a mixture of CMS and CMG complexes. The data suggest that GINS and Sld3 compete with one another for Mcm2-7 and Cdc45 binding. Our results are consistent with a model wherein GINS trades places with Sld3 at a replication origin, contributing to the activation of the replication fork helicase.
authors.affiliation: Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235, USA.
authors.forename: Irina
authors.initials: I
authors.lastname: Bruck
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Kaplan
mesh_terms: D001667:Binding, Competitive; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D005982:Glutathione Transferase / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D018741:Replication Origin; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000378:metabolism*; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D004718:Saccharomycetales / Q000378:metabolism*
chemical_list: C102209:CDC45 protein, S cerevisiae; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C077018:PRP8 protein, S cerevisiae; C513447:PSF2 protein, S cerevisiae; C513448:PSF3 protein, S cerevisiae; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; C474736:SLD5 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C426020:Sld3 protein, S cerevisiae; D005982:Glutathione Transferase; C069199:MCM2 protein, S cerevisiae