Heat induction of a novel Rad9 variant from a cryptic translation initiation site reduces mitotic commitment.
Exposure of human cells to heat switches the activating signal of the DNA damage checkpoint from genotoxic to temperature stress. This change reduces mitotic commitment at the expense of DNA break repair. The thermal alterations behind this switch remain elusive despite the successful use of heat to sensitise cancer cells to DNA breaks. Rad9 is a highly conserved subunit of the Rad9-Rad1-Hus1 (9-1-1) checkpoint-clamp that is loaded by Rad17 onto damaged chromatin. At the DNA, Rad9 activates the checkpoint kinases Rad3(ATR) and Chk1 to arrest cells in G2. Using Schizosaccharomyces pombe as a model eukaryote, we discovered a new variant of Rad9, Rad9-M50, whose expression is specifically induced by heat. High temperatures promote alternative translation from a cryptic initiation codon at methionine-50. This process is restricted to cycling cells and is independent of the temperature-sensing mitogen-activated protein kinase (MAPK) pathway. While full-length Rad9 delays mitosis in the presence of DNA lesions, Rad9-M50 functions in a remodelled checkpoint pathway to reduce mitotic commitment at elevated temperatures. This remodelled pathway still relies on Rad1 and Hus1, but acts independently of Rad17. Heat-induction of Rad9-M50 ensures that the kinase Chk1 remains in a hypo-phosphorylated state. Elevated temperatures specifically reverse the DNA-damage-induced modification of Chk1 in a manner dependent on Rad9-M50. Taken together, heat reprogrammes the DNA damage checkpoint at the level of Chk1 by inducing a Rad9 variant that can act outside of the canonical 9-1-1 complex.
Bangor University, Genome Biology Group, College of Natural Sciences, School of Biological Sciences, Brambell Building, Deiniol Road, Bangor LL57 2UW,  UK.
Simon
S
Janes
Ulrike
U
Schmidt
Karim
K
Ashour Garrido
Nadja
N
Ney
Susanna
S
Concilio
Mohamed
M
Zekri
Thomas
T
Caspari
D000595:Amino Acid Sequence; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000071877:Checkpoint Kinase 1; D004249:DNA Damage; D059565:G2 Phase Cell Cycle Checkpoints; D018869:Heat-Shock Response; D008938:Mitosis; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism*; D010442:Peptide Chain Initiation, Translational; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012568:Schizosaccharomyces / Q000166:cytology* / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000378:metabolism; D013696:Temperature
D018797:Cell Cycle Proteins; D050505:Mutant Proteins; D029702:Schizosaccharomyces pombe Proteins; C069930:rad9 protein; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C000605873:Chk1 protein, S pombe