CDK-dependent complex formation between replication proteins Dpb11, Sld2, Pol ɛ, and GINS in budding yeast

Abstract

Eukaryotic chromosomal DNA replication requires cyclin-dependent kinase (CDK) activity. CDK phosphorylates two yeast replication proteins, Sld3 and Sld2, both of which bind to Dpb11 when phosphorylated. These phosphorylation-dependent interactions are essential and are the minimal requirements for CDK-dependent activation of DNA replication. However, how these interactions activate DNA replication has not been elucidated. Here, we show that CDK promotes the formation of a newly identified fragile complex, the preloading complex (pre-LC) containing DNA polymerase e (Pol e), GINS, Sld2, and Dpb11. Formation of the pre-LC requires phosphorylation of Sld2 by CDK, but is independent of DNA replication, protein association with replication origins, and Dbf4-dependent Cdc7 kinase, which is also essential for the activation of DNA replication. We also demonstrate that Pol e, GINS, Dpb11, and CDK-phosphorylated Sld2 form a complex in vitro. The genetic interactions between Pol e, GINS, Sld2, and Dpb11 suggest further that they form an essential complex in cells. We propose that CDK regulates the initiation of DNA replication in budding yeast through formation of the pre-LC.[Keywords: DNA replication; cell cycle; CDK; GINS; Pol e; yeast] Supplemental material is available at http://www.genesdev.org.

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

“…CDK also phosphorylates Sld3, and phosphorylation of Sld3 by CDK triggers the formation of an Sld3-Dpb11 complex (11,12). CDK-dependent formation of the Sld3-Dpb11 complex may be a mechanism to recruit components of the preloading complex to origins of replication in S phase (10).…”

“…Sld2, Dpb11, GINS, and Pol ⑀ form a transient "preloading complex" that is dependent upon CDK activity (10). CDK also phosphorylates Sld3, and phosphorylation of Sld3 by CDK triggers the formation of an Sld3-Dpb11 complex (11,12).…”

Mcm10 interacts with Rad4/Cut5TopBP1 and its association with origins of DNA replication is dependent on Rad4/Cut5TopBP1

“…In order to establish whether Rad4 TopBP1 and Mcm10 were associated with origins of DNA replication at the point of initiation we synchronised cells in G2 by incubating cdc25-22 mutants, expressing the relevant functional GFP fusion proteins at the wild type locus, at the restrictive temperature and subsequently releasing them at the permissive temperature to undergo synchronous 9 passage through mitosis, septation and initiation of DNA replication. Control Mcm4-GFP associated with three distinct origins of DNA replication, ars1, ars2004 and ars3005, with the peak occurring at 90 min and this pattern was found to be highly reproducible ( with it forming part of the replisome (Fig.…”

“…Upon phosphorylation by CDK, Sld2 and Sld3 interact with distinct BRCT domains of a third protein, Dpb11, the Saccharomyces cerevisiae orthologue of human TopBP1, and that interaction enables recruitment to the pre-RC (6,7,8). Phosphorylation of Sld2 promotes formation of a pre-loading complex containing Dpb11, the heteromeric GINS complex, DNA Pol and Sld2 itself (9). The metazoan orthologue of Sld3, Treslin/Ticrr, has been shown to interact with TopBP1 and to be required for DNA replication (10,11,12).…”

Helicase Loading at Chromosomal Origins of Replication

“…Interestingly, this event occurs at a subset of origins before entry into S phase (Aparicio et al 1999;Kanemaki and Labib 2006;Heller et al 2011;Tanaka et al 2011). S-CDK targets two additional proteins, Sld2 and Sld3, causing them to associate with Dpb11 and eventually resulting in the recruitment of the GINS proteins as well as DNA Pol 1 (Tanaka et al 2007;Zegerman and Diffley 2007;Muramatsu et al 2010). The subsequent activation of DNA unwinding is required for the recruitment of the lagging-strand DNA polymerases (DNA Pol a-primase and DNA Pol d (Mimura et al 2000;Heller et al 2011).…”