Rad53 checkpoint kinase phosphorylation site preference identified in the Swi6 protein of Saccharomyces cerevisiae.
Rad53 of Saccharomyces cerevisiae is a checkpoint kinase whose structure and function are conserved among eukaryotes. When a cell detects damaged DNA, Rad53 activity is dramatically increased, which ultimately leads to changes in DNA replication, repair, and cell division. Despite its central role in checkpoint signaling, little is known about Rad53 substrates or substrate specificity. A number of proteins are implicated as Rad53 substrates; however, the evidence remains indirect. Previously, we have provided evidence that Swi6, a subunit of the Swi4/Swi6 late-G(1)-specific transcriptional activator, is a substrate of Rad53 in the G(1)/S DNA damage checkpoint. In the present study we identify Rad53 phosphorylation sites in Swi6 in vitro and demonstrate that at least one of them is targeted by Rad53 in vivo. Mutations in these phosphorylation sites in Swi6 shorten but do not eliminate the Rad53-dependent delay of the G(1)-to-S transition after DNA damage. We derive a consensus for Rad53 site preference at positions -2 and +2 (-2/+2) and identify its potential substrates in the yeast proteome. Finally, we present evidence that one of these candidates, the cohesin complex subunit Scc1 undergoes DNA damage-dependent phosphorylation, which is in part dependent on Rad53.
Basic Sciences Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N., Seattle, WA 98109, USA.
Julia M
JM
Sidorova
Linda L
LL
Breeden
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins; D002455:Cell Division; D064447:Checkpoint Kinase 2; D004249:DNA Damage; D016193:G1 Phase; D005982:Glutathione Transferase / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D020543:Proteome; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016196:S Phase; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D018797:Cell Cycle Proteins; D010748:Phosphopeptides; D020543:Proteome; D011993:Recombinant Fusion Proteins; C075111:SWI6 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D005982:Glutathione Transferase; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae