Download Checkpoints

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