SUMO-binding motifs mediate the Rad60-dependent response to replicative stress and self-association.
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.
Raffa
Grazia D
GD
Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Wohlschlegel
James
J
Yates
John R
JR
Boddy
Michael N
MN
D020816:Amino Acid Motifs; D001665:Binding Sites; D064447:Checkpoint Kinase 2; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry* / Q000502:physiology; D004260:DNA Repair; D004261:DNA Replication; D019281:Dimerization; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry; D025842:SUMO-1 Protein / Q000378:metabolism*; D012568:Schizosaccharomyces; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry* / Q000502:physiology
D002868:Chromosomal Proteins, Non-Histone; C455501:Rad60 protein, S pombe; D025842:SUMO-1 Protein; D029702:Schizosaccharomyces pombe Proteins; D064447:Checkpoint Kinase 2; C578634:CHEK2 protein, human; C578636:Cds1 protein, S pombe; D017346:Protein Serine-Threonine Kinases