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Molecular and Cellular Biology, May 2003, p. 3405-3416, Vol. 23, No. 10
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.10.3405-3416.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Rad53 Checkpoint Kinase Phosphorylation Site Preference Identified in the Swi6 Protein of Saccharomyces cerevisiae

Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109

Received 26 September 2002/ Returned for modification 5 November 2002/ Accepted 19 February 2003

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-G₁-specific transcriptional activator, is a substrate of Rad53 in the G₁/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 G₁-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.

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