Rad3-dependent phosphorylation of the checkpoint clamp regulates repair-pathway choice.
When replication forks collapse, Rad3 phosphorylates the checkpoint-clamp protein Rad9 in a manner that depends on Thr 225, a residue within the PCNA-like domain. The physiological function of Thr 225-dependent Rad9 phosphorylation, however, remains elusive. Here, we show that Thr 225-dependent Rad9 phosphorylation by Rad3 regulates DNA repair pathways. A rad9(T225C) mutant induces a translesion synthesis (TLS)-dependent high spontaneous mutation rate and a hyper-recombination phenotype. Consistent with this, Rad9 coprecipitates with the post-replication repair protein Mms2. This interaction is dependent on Rad9 Thr 225 and is enhanced by DNA damage. Genetic analyses indicate that Thr 225-dependent Rad9 phosphorylation prevents inappropriate Rhp51-dependent recombination, potentially by redirecting the repair through a Pli1-mediated sumoylation pathway into the error-free branch of the Rhp6 repair pathway. Our findings reveal a new mechanism by which phosphorylation of Rad9 at Thr 225 regulates the choice of repair pathways for maintaining genomic integrity during the cell cycle.
Kai
Mihoko
M
Department of Pathology, Stanford University School of Medicine, Stanford, California 94305-5324, USA.
Furuya
Kanji
K
Paderi
Francesca
F
Carr
Antony M
AM
Wang
Teresa S F
TS
D002453:Cell Cycle / Q000235:genetics; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D064447:Checkpoint Kinase 2; D004249:DNA Damage / Q000235:genetics; D004260:DNA Repair / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal / Q000235:genetics; D018816:Genes, cdc / Q000502:physiology*; D042822:Genomic Instability / Q000235:genetics*; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics; D013912:Threonine / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism
D018797:Cell Cycle Proteins; D029702:Schizosaccharomyces pombe Proteins; C069930:rad9 protein; D013912:Threonine; C513353:Mms2 protein, S pombe; D044763:Ubiquitin-Conjugating Enzymes; C063863:rhp6 protein, S pombe; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C456840:rad3 protein, S pombe