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

Abstract

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.

ACKNOWLEDGMENTS

We gratefully acknowledge members of the Breeden lab for support and numerous discussions. We particularly thank Bernard Mai for generating pBD1998; Steve Elledge for helpful discussions and for providing baculovirus-expressed Rad53 and anti-Rad53 serum; and Andrew Emili, Robert Sclafani, and Sue Biggins for strains and constructs.

This work was supported by the NIH grant GM41073 to L.L.B. and by a Leukemia and Lymphoma Society senior fellowship to J.M.S.