CDK-dependent phosphorylation of Sld2 and Sld3 initiates DNA replication in budding yeast

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Abstract
This paper, one of two published that show Sld2 and Sld3 constitute the minimal group of cyclin-dependent kinase (CDK) targets necessary to promote replication, also finds that the function of G1 CDKs in replication is not just to activate the S-phase CDKs, but also to regulate the Cdc7 kinase regulatory subunit, Dbf4. In eukaryotic cells, cyclin-dependent kinases (CDKs) have an important involvement at various points in the cell cycle. At the onset of S phase, active CDK is essential for chromosomal DNA replication1, although its precise role is unknown. In budding yeast (Saccharomyces cerevisiae), the replication protein Sld2 (ref. 2) is an essential CDK substrate3, but its phospho-mimetic form (Sld2-11D) alone neither affects cell growth4 nor promotes DNA replication in the absence of CDK activity4, suggesting that other essential CDK substrates promote DNA replication. Here we show that both an allele of CDC45 (JET1) and high-copy DPB11, in combination with Sld2-11D, separately confer CDK-independent DNA replication. Although Cdc45 is not an essential CDK substrate, CDK-dependent phosphorylation of Sld3, which associates with Cdc45 (ref. 5), is essential and generates a binding site for Dpb11. Both the JET1 mutation and high-copy DPB11 by-pass the requirement for Sld3 phosphorylation in DNA replication. Because phosphorylated Sld2 binds to the carboxy-terminal pair of BRCT domains in Dpb11 (ref. 4), we propose that Dpb11 connects phosphorylated Sld2 and Sld3 to facilitate interactions between replication proteins, such as Cdc45 and GINS. Our results demonstrate that CDKs regulate interactions between BRCT-domain-containing replication proteins and other phosphorylated proteins for the initiation of chromosomal DNA replication; similar regulation may take place in higher eukaryotes.