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RAD genes rad mutants are hypersensitive to DNA damaging agents X-irradiation UV DNA synthesis inhibitors Possible RAD gene functions Recognize DNA damage and catalyze its repair Recognize DNA damage and activate a checkpoint mechanism that arrests the cell cycle to enable time for repair Irradiation causes cell cycle delay MBC treatment rescues radiation-induced lethality hold in MBC eni wildtype rad9 Diamond: x-ray alone Square: arrest with MBC, release and X-ray Triangle: arrest with MBC, x-ray and hold in MBC for 4 hr rad52 DNA synthesis mutants lose viability in rad9 background Use this phenotype to look for new checkpoint mutants 16,000 mutagenized colonies 500 die rapidly 21 fail to arrest Define 5 genes Checkpoint mutants are sensitive to a variety of DNA damaging agents As expected, checkpoint mutants are indeed defective for cell cycle arrest following irradiation Some G2-phase checkpoint mutants are also defective for an S-phase checkpoint Model Rad53 is phosphorylated in response to DNA damage. Phosphorylation depends on Mec1 but not Rad9 Phosphoforms * Removed by phosphatase * * No phoshoforms in mec1 mutant Conservation of checkpoint genes – complementation of rad53 by human CHK2 human Dros pombe cerev. Human Chk2 is modified in response to irradiation Panels A and B use Ab to identify Chk2 protein Panels C and D show Chk2 mobility shift in response to irradiation Activation of Chk2 is ATM (Mec1) dependent Mre11/Xrs2/Rad50 complex IP with Ab to Mre11 Test with Ab to others * IP with Ab to Xrs2 Test with Ab to others * Mre11 is required for phosphorylation of Rad53 in response to X-irradiation The Mre11/Rad50/Xrs2 complex is required for phosphorylation of Rad53 in response to X-irradiation Roles of ATM and ATR in G2 checkpoint activation. (Mec1) (Rad53) (Regulator of Cdc2) Abraham R T Genes Dev. 2001;15:2177-2196 ©2001 by Cold Spring Harbor Laboratory Press