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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 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 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 8  Autophagy contributes to Sae2 degadation, perhaps through acetylation

5. Gcn5, Rpd3 & Hda1 control Sae2 level 9 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 inhibition d. Gcn5 HAT counteracts Rpd3 & Hda1 activity

… Gcn5, Rpd3 & Hda1 control Sae2 level 10 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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