Post on 29-Dec-2015
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IntroductionIntroduction1. 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
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IntroductionIntroduction
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
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1. VPA counteracts the DNA damage reponse1. VPA counteracts the DNA damage reponse
VPA (valproic acid): HDAC inhibitorHO: a yeast nuclease that regconizes a specific DNA sequence- Rad53 activation needs 10 kb of ssDNA
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… 1. VPA counteracts the DNA damage reponse… 1. VPA counteracts the DNA damage reponse
c. VPA counteractes Pfa1 & Ddc recruitment d. VPA reduces ectopic recombination (rec.)
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2. Sae2 & Exo1 are degraded in VPA-treated cells2. Sae2 & Exo1 are degraded in VPA-treated cells
VPA-induced DSB resection defects both Sae2 & Exo1 VPA-induced DSB resection defects both Sae2 & Exo1
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3. VPA stimulates autophagy?3. VPA stimulates autophagy?
Autophagy induction correlates w/t:1. Vascuolar staining of Cherry-
Ape1 to perivacuolar foci & GFP-Atg8
2. Pho8∆60 (autophagy marker)3. Processing of GFP-Atg8
ATG1: encodes an main autophagy kinase
VPA induces autophagy VPA induces autophagy
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4. VPA-induced Sae2 acetylation & degradation4. 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
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Autophagy contributes to Sae2 degadation, perhaps through acetylation
Autophagy contributes to Sae2 degadation, perhaps through acetylation
5. Gcn5, Rpd3 & Hda1 control Sae2 level5. Gcn5, Rpd3 & Hda1 control Sae2 level
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a. 4NQO: an ultraviolet-mimetic drug HU: a DNA synthesis inhibitor MMS: a DNA-akylating agent
a. 4NQO: an ultraviolet-mimetic drug HU: a DNA synthesis inhibitor MMS: a DNA-akylating agent
c. Hda1& hda1 mutants = HDAC inhibitionc. Hda1& hda1 mutants = HDAC inhibition
d. Gcn5 HAT counteracts Rpd3 & Hda1 activity
d. Gcn5 HAT counteracts Rpd3 & Hda1 activity
… Gcn5, Rpd3 & Hda1 control Sae2 level… Gcn5, Rpd3 & Hda1 control Sae2 level
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e. Rapamycin: stimulates Autophagy f. Camptothecin (CPT) inhibits the DNA enzyme
topoisomerase I
e. Rapamycin: stimulates Autophagy f. Camptothecin (CPT) inhibits the DNA enzyme
topoisomerase I
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