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.The Mcm2-7 complex functions with Cdc45 and the foursubunit GINS complex (composed of Psf1, Psf2, Psf3, and Sld5) to unwind DNA at a replication fork in eukaryotic organisms (1-3). The Mcm2-7 complex exhibits weak helicase activity on its own, but it is stimulated by the presence of Cdc45 and GINS, suggesting that Cdc45 and GINS function as helicase-stimulating factors (2, 4). The Mcm2-7, Cdc45, and GINS proteins are all essential for cell growth, suggesting that their function in unwinding DNA at a replication fork is required for DNA replication.Sld3 is also essential for cell growth, but the role of Sld3 in DNA replication may be limited to the initiation step. Whereas the Mcm2-7, Cdc45, and GINS proteins travel with the replication fork, Sld3 does not travel with the fork (5). Sld3 forms a complex with Cdc45 in vivo, and Sld3 is required for the association of Cdc45 with origins or replication (6). The mechanism of how Sld3 triggers the initiation of DNA replication is not well understood.The loading of DNA replication proteins as a function of cell cycle is beginning to be elucidated. In late M phase and early G 1 ,

Origin Single-stranded DNA Releases Sld3 Protein from the Mcm2–7 Complex, Allowing the GINS Tetramer to Bind the Mcm2–7 Complex

“…We next found that Sld3 can form an interaction with Mcm2-7 with 1:1 stoichiometry in the absence of DNA and that GINS can form an interaction with Mcm2-7 with 1:1 stoichiometry in the absence of DNA (Fig. 8, D-F) (39). However, in the presence of ssARS1-2, the interaction between Sld3 and Mcm2-7 is disrupted (Fig.…”

“…When Sld3 is added to a GST-Mcm2-7 pulldown of GINS, Sld3 inhibits the interaction between GINS and Mcm2-7 (Fig. 7, C and D) (39). When ssARS1-2 is added to the GST-Mcm2-7 pulldown of radiolabeled GINS in the presence of competing Sld3, ssARS1-2 promotes the interaction between GINS and Mcm2-7 in a concentration-dependent manner (Fig.…”

“…As a control, Sld3 does not bind directly to GINS (Fig. 8, A and B) (39). Cdc45 and Sld3 bind together in vitro (39) and in vivo (18), and thus, we also performed these experiments in the presence of Cdc45.…”

“…8, A and B) (39). Cdc45 and Sld3 bind together in vitro (39) and in vivo (18), and thus, we also performed these experiments in the presence of Cdc45. When ssARS1-2 is added to the GSTMcm2-7 pulldown of radiolabeled GINS in the presence of Cdc45 and Sld3, ssARS1-2 stimulates the interaction between GINS and Mcm2-7 in a concentration-dependent manner (Fig.…”

A Positive Amplification Mechanism Involving a Kinase and Replication Initiation Factor Helps Assemble the Replication Fork Helicase

“…Human TopBP1 was purified as described previously (49). Plasmids for E. coli recombinant expression of Dpb11 (wild-type and mutant) for GINS and for yeast in vivo expression of DPB11 (wild-type and mutants) were prepared as described previously (37,50). Yeast Dpb11 (wild-type and mutant), Mcm2-7, Cdc45, Sld3, Sld2, CDK, DDK, GINS, and GST were purified as described previously (37,46,48,51).…”