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

Irina Bruck,Daniel L Kaplan

doi:10.1074/jbc.m111.226332

Abstract

The replication fork helicase in eukaryotic cells is comprised of Cdc45, Mcm2-7, and GINS (CMG complex). In budding yeast, Sld3, Sld2, and Dpb11 are required for the initiation of DNA replication, but Sld3 and Dpb11 do not travel with the replication fork. Sld3 and Cdc45 bind to early replication origins during the G(1) phase of the cell cycle, whereas Sld2, GINS, polymerase ε, and Dpb11 form a transient preloading complex that associates with origins during S phase. We show here that Sld3 binds tightly to origin single-stranded DNA (ssDNA). CDK-phosphorylated Sld3 binds to origin ssDNA with similar high affinity. Origin ssDNA does not disrupt the interaction between Sld3 and Dpb11, and origin ssDNA does not disrupt the interaction between Sld3 and Cdc45. However, origin ssDNA substantially disrupts the interaction between Sld3 and Mcm2-7. GINS and Sld3 compete with one another for binding to Mcm2-7. However, in a mixture of Sld3, GINS, and Mcm2-7, origin ssDNA inhibits the interaction between Sld3 and Mcm2-7, whereas origin ssDNA promotes the association between GINS and Mcm2-7. We also show that origin single-stranded DNA promotes the formation of the CMG complex. We conclude that origin single-stranded DNA releases Sld3 from Mcm2-7, allowing GINS to bind Mcm2-7.

Highlights

In addition to Mcm2–7, the replication fork helicase contains the accessory factor Cdc45 and the GINS complex [12,13,14]
In a mixture of Sld3, GINS, and Mcm2–7, origin single-stranded DNA (ssDNA) inhibits the interaction between Sld3 and Mcm2–7, whereas origin ssDNA promotes the association between GINS and Mcm2–7
Origin Single-stranded DNA Releases Sld3 from Mcm2–7, Allowing GINS to Bind Mcm2–7—We examined whether ssARS1-2 affects the interaction between GSTMcm2–7 and GINS (Fig. 7, A and B)

Summary

Introduction

In addition to Mcm2–7, the replication fork helicase contains the accessory factor Cdc45 and the GINS complex [12,13,14]. Biotinylated ssARS1-4 or dsARS1-12 did not pull down a high fraction of input PKA or CDK-radiolabeled Sld3, confirming that Sld3 interaction with origin DNA is region- and structure-specific. Origin ssDNA (ssARS1-2) Disrupts Interaction between Sld3 and Mcm2–7—Sld3 binds to Dpb11 in vivo when it is phosphorylated by CDK [24, 25].

Results

Conclusion