GINS and Sld3 Compete with One Another for Mcm2-7 and Cdc45 Binding

Irina Bruck,Daniel L Kaplan

doi:10.1074/jbc.m111.218305

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.

Highlights

Sld3 is essential for cell growth, but the role of Sld3 in DNA replication may be limited to the initiation step
We found that Sld3 and Cdc45 form a stable complex that is independent of cyclin-dependent kinase (CDK) activity
When Sld3, Cdc45, GINS, and Mcm2-7 Are Added Together, the Result Is a Mixture of CMG and CMS Complexes—We found that the addition of Cdc45, Mcm2-7, and Sld3 results in a CMS complex with 1:1:1 stoichiometry (Fig. 2), and the addition of Cdc45, Mcm2-7, and GINS results in a CMG complex with a 1:1:1 stoichiometry (Fig. 5)

Summary

Introduction

Sld3 is essential for cell growth, but the role of Sld3 in DNA replication may be limited to the initiation step. We radiolabeled Cdc45 and subjected the protein to size exclusion chromatography in the presence or absence of Sld3 (Fig. 2A). As the concentration of Sld3 was increased in this reaction, there was a progressive decrease in the interaction observed between GST-Cdc45 and radiolabeled GINS (Fig. 4, E and F).

Results

Conclusion