Download 슬라이드 1

1
Introduction
1.
DNA damage checkpoint response includes ATR & ATM
+ ATR (ataxia telangiectasia and Rad3-related protein) is a serine/threonine-specific protein kinase that is involved in
sensing DNA damage and activating the DNA damage checkpoint, leading to cell cycle arrest.
- ATR is assisted by Ddc2
- Rad53 (CHK2 in humans) protein kinase
+ ATM (Ataxia telangiectasia mutated) is a serine/threonine-specific protein kinase that is recruited and activated by
DNA double-strand breaks. It phosphorylates several key proteins that initiate activation of the DNA damage
checkpoint, leading to cellcycle arrest, DNA repair or apotosis
2
Introduction
2. Autophagy: the degradation of intracellular components via the
lysosome. It’s triggered by Nutrient starvation and TOR1 inhibitiors
3. Histone Acetyltransferase (HAT): Gcn5.
Histone Deacetylase (HDAC): Hda1 & Rpd3
3
1. VPA counteracts the DNA damage reponse
VPA (valproic acid): HDAC inhibitor
HO: a yeast nuclease that regconizes a specific
DNA sequence
- Rad53 activation needs 10 kb of ssDNA
4
… 1. VPA counteracts the DNA damage reponse
c. VPA counteractes Pfa1 & Ddc recruitment
d. VPA reduces ectopic recombination (rec.)
5
2. Sae2 & Exo1 are degraded in VPA-treated cells
 VPA-induced DSB resection defects both Sae2 & Exo1
6
3. VPA stimulates autophagy?
Autophagy induction correlates w/t:
1. Vascuolar staining of CherryApe1 to perivacuolar foci & GFPAtg8
2. Pho8∆60 (autophagy marker)
3. Processing of GFP-Atg8
ATG1: encodes an main autophagy
kinase
 VPA induces autophagy
7
4. VPA-induced Sae2 acetylation & degradation
a. 1:input Sae2
2. Sae2-HA –VPA
3. As in 2 + VPA
4. 3ul elution Sae2-HA –VPA after immunoprecipitation (IP, input Ack-IP)
5. Double amount of 4
6. IP anti-AcK elution from anti-HA IP of Sae2-HA –VPA
7. As in 6 but + VPA
- Anti-HA & anti-acetyl-lysine antibodies
b. PMSF blocks autophagy
d. Rapamycin: induces autophagy by inhibiting Tor1
 Autophagy contributes to Sae2
degadation, perhaps through acetylation
8
5. Gcn5, Rpd3 & Hda1 control Sae2 level
a. 4NQO: an ultraviolet-mimetic drug
HU: a DNA synthesis inhibitor
MMS: a DNA-akylating agent
c. Hda1& hda1 mutants = HDAC inhibition
d. Gcn5 HAT counteracts Rpd3 & Hda1 activity
9
… Gcn5, Rpd3 & Hda1 control Sae2 level
e. Rapamycin: stimulates Autophagy
f. Camptothecin (CPT) inhibits the DNA enzyme
topoisomerase I
10
11