1. 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

2. Irradiation causes cell cycle delay

3. eni MBC treatment rescues radiation-induced lethality wildtyperad9 rad52 hold in MBC 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

4. 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

5. Checkpoint mutants are sensitive to a variety of DNA damaging agents

6. As expected, checkpoint mutants are indeed defective for cell cycle arrest following irradiation

7. Some G2-phase checkpoint mutants are also defective for an S-phase checkpoint

8. Model

9. Rad53 is phosphorylated in response to DNA damage. Phosphorylation depends on Mec1 but not Rad9 Phosphoforms * Removed by phosphatase * No phoshoforms in mec1 mutant *

10. Conservation of checkpoint genes – complementation of rad53 by human CHK2 human Dros pombe cerev.

11. 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

12. Activation of Chk2 is ATM (Mec1) dependent

13. IP with Ab to Mre11 Test with Ab to others IP with Ab to Xrs2 Test with Ab to others Mre11/Xrs2/Rad50 complex * *

14. Mre11 is required for phosphorylation of Rad53 in response to X-irradiation

15. The Mre11/Rad50/Xrs2 complex is required for phosphorylation of Rad53 in response to X-irradiation

16. Roles of ATM and ATR in G2 checkpoint activation. Abraham R T Genes Dev. 2001;15:2177-2196 ©2001 by Cold Spring Harbor Laboratory Press (Mec1) (Rad53) (Regulator of Cdc2)