Role of Rad9 cell cycle phosphorylation in the regulation of Chk1 activation

DNA damage checkpoints are triggered in response to DNA insults. Once activated, these pathways prevent replication and segregation of the damaged DNA. In addition to cell cycle delays they regulate repair, transcription and the apoptotic response to DNA damage. Budding yeast RAD9 was the first checkpoint gene identified. The checkpoint activity of this protein has been reported in all phases of the cell cycle. Loss of this gene impairs checkpoint-activated cell cycle arrests and increases genomic instability. One of its important function is to regulate the two downstream effector kinases Rad53 and Chk1. Rad9 is phosphorylated in normal cell cycle and hyperphosphorylated in response to DNA damage. Genetic analyses indicate that cell cycle phosphorylation of Rad9 is Cdc28/Clb dependent. The protein contains 20 potential cdk phosphorylation sites (S/T-P-X-K/R) and 9 of them are clustered in Nterminal region that has been shown to be necessary for Chk1 activation. The aim of this study was to identify a role for the N-terminal CDK phosphorylation sites in the regulation of Chk1. To do this novel rad9 mutants were designed, generated and analysed. In particular, they were characterized for their ability to activate Chk1. We have found that the nine putative CDK phosphorylation sites of the Rad9 CAD region is absolutely required for damage induced Chk1 activation in G2/M cells. Based on the yeast two hybrid and immunoprecipitation interaction studies, these sites are required for the constitutive interaction between Rad9 and Chk1. Cdc28 activity is required for their interaction in G2/M but not in G1 cells. We have identified a subset of sites necessary for Chk1 activation in response to DNA damage. Out of nine sites CDK6 and CDK7 are the only required for a Cdc28- dependent interaction between Rad9 and Chk1. Our data suggests a model where Rad9 and Chk1 interact constitutively, with remodeling of this complex in response to DNA damage requiring Mec1-dependent phosphorylation