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Abstract
In fission yeast, the replication checkpoint is enforced by the kinase Cds1 (human Chk2), which regulates both cell cycle progression and DNA repair factors to ensure that the genome is faithfully duplicated prior to mitosis. Cds1 contains a forkhead-associated domain that mediates its interaction with phosphorylated residues in target proteins. One target of Cds1 is the essential nuclear protein Rad60, which contains the unique structural feature of tandem SUMO homology domains at its C terminus. Hypomorphic mutants of Rad60 cause profound defects in DNA repair and replication stress tolerance. To explore the physiological significance of the Cds1-Rad60 interaction, we have examined the phosphorylation of Rad60 by Cds1 in vitro and the in vivo phosphorylation of Rad60 in response to replication blocks. We find that the N terminus but not the SUMO-like domain of Rad60 is phosphorylated in both conditions. Three important Rad60 phosphorylation sites were identified: Thr(72), Ser(32), and Ser(34). Rad60 Thr(72) mediates the Cds1-Rad60 interaction and is required for the Cds1-dependent phosphorylation of Rad60 in response to replication arrest. Phosphorylation of Rad60 Ser(32) and Ser(34) in a putative SUMO-binding motif is critical for the survival of replication stress. In addition, mutation of Rad60 Ser(32) and Ser(34) to alanine is lethal in cells deleted for the RecQ DNA helicase Rqh1. Finally, we find that Rad60 self-associates via its C-terminal SUMO-like domain and putative SUMO-binding motifs.
Highlights
Genome integrity is threatened by multiple sources of damage throughout the cell cycle
The protein encoded by another hypomorphic allele of rad[60], rad[60-4] (T72A/I232S/Q250R/K312N) fails to interact with Cds[1] and does not undergo the typical Cds1-dependent phosphorylation when replication is blocked (2). rad60-4-expressing cells are highly sensitive to HU but only moderately sensitive to UV and do not display any vegetative growth defects (2)
We have explored the functional relationship between the replication checkpoint kinase Cds[1] and the recombination repair factor Rad[60]
Summary
Genome integrity is threatened by multiple sources of damage throughout the cell cycle. Rad[60] Thr[72] mediates the Cds1Rad[60] interaction and is required for the Cds1-dependent phosphorylation of Rad[60] in response to replication arrest. One such factor is the homologous recombination repair protein Rad[60], which is regulated via direct interaction with the replication checkpoint kinase Cds[1] (2, 3).
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