Heat induction of a novel Rad9 variant from a cryptic translation initiation site reduces mitotic commitment

Abstract

Exposure of human cells to heat switches DNA damage signaling 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 sensitize 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 Rad3ATR and Chk1 to arrest cells in G2. Using Schizosaccharomyces pombe as a model eukaryote, we discovered a new variant of Rad9, Rad9-M50, expression of which 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 independent of the temperature-sensing MAP kinase pathway. While full-length Rad9 delays mitosis in the presence of DNA lesions, Rad9-M50 functions in a remodeled checkpoint pathway to reduce mitotic commitment at elevated temperatures. This remodeled pathway still relies on Rad1 and Hus1, but acts independently of Rad17. Heat-induction of Rad9-M50 ensures that Chk1 kinase 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 reprograms 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.