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| CELL CYCLE: Why phosphorylation matters
There is evidence in budding yeast that the replication protein Sld2 is phosphorylated in a cyclin-dependent kinase (CDK)-dependent manner, and that it is essential for chromosomal replication. One of the puzzles in S-phase biology is that although phosphorylation of replication proteins is generally believed to activate DNA replication, finding the biological effects of phosphorylation has been difficult to prove. But, reporting in Nature, Araki and colleagues provide several lines of evidence in budding yeast that the replication protein Sld2 is phosphorylated in a cyclin-dependant kinase (CDK) manner and that this is essential for chromosomal DNA replication. The authors looked at Sld2 — which is required for an early step of chromosomal DNA replication — as it has six preferred CDK phosphorylation motifs, and mutations in any one of these motifs are lethal. Araki and colleagues confirmed that Sld2 was phosphorylated by carrying out mobility-shift analysis of Sld2. Lower-migrating forms of Sld2 appeared from early S phase, mostly disappeared before the subsequent S phase, and were abolished by phosphatase treatment. In vitro kinase assays suggested that Sld2 is phosphorylated by activity of S-phase CDKs, as Sld2 was phosphorylated by p13SUC1-bound protein kinases (p13SUC1 specifically binds to Cdc28 — the catalytic subunit of the Cdks in budding yeast) and purified human CDK kinase. Inhibiting the S-Cdk kinase activity in cells and creating cells that lacked the S-phase cyclins Clb5 and Clb6, suggested a similar phosphorylation mechanism occurs in vivo. But is this phosphorylation important for cell growth and chromosomal DNA replication? To examine this, the authors created cells that contained mutations in the preferred CDK phosphorylation motifs. They found that alleles containing mutations in each of six sites could substitute for the wild-type gene, whereas the mutations in all six sites (All-A) could not, showing that the individual motifs are functionally redundant. All-A mutant protein levels were similar to wild-type and single mutant proteins, but in wild-type cells, the All-A protein did not show a mobility shift in S phase. This suggests that S-Cdk-dependent phosphorylation of Sld2 is essential for cell growth. For chromosomal DNA replication to occur, Sld2 forms a complex with Dpb11, so the authors looked at whether phosphorylation of Sld2 is key to this process. Immunoprecipitation studies showed that the Sld2–Dpb11 complex is formed predominantly in S-phase. Removing phosphatase inhibitors from the cell extract or introducing the All-A mutant instead of Sld2 prevented a complex from forming. Increased levels of Dpb11 suppressed the growth defect of the All-A mutants as expected from its defect of the complex formation (all the SLD2 mutations defective in the complex formation were suppressed by the increased levels of DPB11). So Araki and colleagues say this shows that phosphorylation of Sld2 is essential for chromosomal DNA replication. A similar situation could occur in fission yeast, as phosphorylation of drc1, a possible homologue of Sld2, also seems to be essential for cell growth and DNA replication. Simon Frantz References
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