Abstract
Cyclin-dependent kinases (CDKs) regulate the progression of the cell cycle in eukaryotes. One of the major roles of CDK is to promote chromosomal DNA replication. However, how CDKs promote DNA replication has been a long-standing question, because all the essential CDK substrates in DNA replication have not been identified yet. Recently Sld2 and Sld3 were identified as essential substrates of CDKs in the initiation step of DNA replication in budding yeast. Moreover, bypass of their phosphorylations is sufficient to promote DNA replication. Phosphorylation of Sld2 and Sld3 by CDKs enhances the formation of complex(es) with a BRCT (BRCA1 C-Terminal)-containing replication protein, Dpb11. We further propose that multiple phosphorylation by CDKs controls this process in budding yeast. Even though Sld3 orthologues in multicellular eukaryotes have not been identified, similar complex formation and, therefore, a similar mechanism of initiation control might be employed in eukaryotes.
Highlights
It has been known for a long time that Cyclin-dependent kinases (CDKs) activity is required for DNA replication in eukaryotes
CDKcontaining fractions activated the replication of SV40 virus in cellular extracts [1] ; Immunodepletion of CDKs prevented the initiation of DNA replication in vitro in Xenopus egg extracts [2]; and CDK or cyclin mutants cannot promote DNA replication in yeasts [3,4]
We and others recently revealed that phosphorylation of two replication proteins, Sld2 and Sld3, is essential and minimal requirement for CDK-dependent activation of the initiation of DNA replication in budding yeast (Fig. 1) [5,6]
Summary
It has been known for a long time that CDK activity is required for DNA replication in eukaryotes. The fusion protein between Sld3-2A and C-terminal half of Dpb (SD fusion) can replace Sld and Dpb simultaneously because N-terminal pair of Dpb functions as the binding site to Sld and SD fusion functions as Sld3-Dpb complex[5] These results imply that phosphorylation-dependent interaction between Dpb and Sld is CDK-regulated essential step for the initiation of DNA replication. Semi-conservative DNA synthesis initiates from known replication origins in a pre-RC-dependent manner with proper replication fork complex, CMG (Cdc45-Mcm-GINS) in JET1-1 sld2-D mutant cells lacking CDK activity (DNA synthesis in SD fusion strain has not been characterized in detail). Efficient binding of Sld to N-terminal pair of BRCT domains in Dpb requires simultaneous phosphorylations of Thr600 and Ser622, which may require high activity of Cdk. This evidence suggests that plant cells employ phosphorylation-dependent interaction different from BRCT phosphopeptide domains
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