Week 4: DNA-PKcs And ATM Supress DNA End Resection

Question 1

PIKKs summary

Answer 1

1. DSB->Ku->DNAPKcs->NHEJ
2. DSB->MRN->ATM->P-CHK2->P-H2AX->mdc1->HR or NHEJ
3. SSB->RPA->ATRIP->ATR
   3a. ATR->p-chk1
   3b. ATR->p-RPA->Nbs1

Question 2

What are the two unique characteristics of the design of this study

Answer 2

1. It examined kinase crosstalk as a function of DSB load
2. It analyzed only in the G2 phase of the cell cycle

Question 3

What did these experiments show

Answer 3

1. At low IR doses, ATM and ATR regulate epistemically as a module at the G2 checkpoint
2. At high IR doses, DNA-PKcs also works w ATM/ATR and causes hyper-resection at G2 checkpoint activation

Question 4

Figure 1A/B

Answer 4

1. Objective: to see what is essential for manifestation of G2 checkpoint in S phase irradiated cells
2. What they did: PI stain & flow cytometry. Exposed hTert (a) and LLC (b) to low IR dose (2Gy, 4Gy) to impair cell division
3. Results
   2a. Untreated cells + Chk2 inhibitor cells + ATM inhibitor =high cells in G2=checkpoint works
   2b. NI (no IR) , ATRi, Chk1i: causes suppression of G2 checkpoint (ATR activates Chk1)=bye bye checkpoint
4. Conclude G2 checkpoint activated in S phase cells exposed to low IR doses is under complete control of ATR (NEED ATR for G2 checkpoint)

Question 5

Figure 1C

Answer 5

1. Objective: to confirm effects noted in 1b/ validate the results in S phase cells
2. What they did: pulse labelled A549 (LLC) cells with BrdU (Arrest in S phase) just before IR (4Gy)
3. Results: shows treatment with ATRi removes the G2 checkpoint in cells exposed to 4 Gy (NEED ATR for G2 checkpoint)

Question 6

Figure 1D

Answer 6

1. Objective: to confirm the full control of ATR on G2 checkpoint via western
2. -DOX: regular ATR expression; +DOX: ATR expression that is catalytically dead (Can’t be activated; which is why it’s lower)
3. Results: validation for figure 1e/f saying that DOX does its job

Question 7

Figure 1E/F

Answer 7

1. Objective: used ATRkd cell line w 4Gy in S phase comparing no DOX (e) and DOX (f)
2. -DOX: regular ATR activity; which is why untreated is arrested (ATR is functional)
3. +DOX: ATR is nonfunctional; which is why untreated doesn’t arrest anymore (ATR can’t do its job)

Question 8

Figure 3a/b/d

Answer 8

1. Figure 3a vs b: DNA-Pkcs deficient (M059J) cells have a stronger arrest in G2 than DNA-Pkcs proficient cells (M059K)
   1a. shows us the hyperactivated G2 checkpoint relies fully on ATR and Chk1
   1b. also shows that DNA-pkcs is required to supress checkpoint hyperactivation
2. Figure 3a vs 3d: higher IR (d)=higher checkpoint activation

Question 9

Figure 3c

Answer 9

1. hTert cells with DNA-PKcs proficiency in low IR impacts the G2 checkpoint cells using combination inhibitors (DNAPKcsi, DNAPKcsi+ ATMi, and DNAPKcsi + ATRi + ATMi).
2. Results: when ATR inhibitor introduced, cells aren’t in G2 arrest (need ATR to do arrest)

Question 10

Figure 3e/f

Answer 10

1. Objective: assessed hyperactivation of ATR w and w/o IR, in cells that are either DNA-PKcs proficient, DNA-PKcs deficient, ATM proficient, or ATM deficient via looking at p-Chk1
2. Figure 3e: high ATR activation (high Chk1) in DNA-Pkcs deficient cells, when doses of radiation are higher versus not (high IR=high damage=more pChk1 (damage response))
3. figure 3f: when ATRi, Chk1 doesn’t get phosphorylated and when DNA-PKcsi there is a phosphorylation of CHK1 (small); and when no inhibitors, strongest phosphorylation of chk1 (DNA-PKcs diminishes ATR signalling)

Question 11

Figure 5A/B/C

Answer 11

1. Objective: to detect if ATR plays a role in DNA end resection (RPA)
2. EDU labels cells in S; resection is measured when RPA is in EDU+ cells
3. Results (a): higher IR=higher RPA signal=higher resection at DSB
4. Results (b): shows IR induced resection via ATR can be quantified in a range of doses between 5-15 Gy
5. Results (c): ATRi has little effect on overall IR induced resection

Question 12

Figure 5D

Answer 12

1. Inhibition of ATR by DOX (removes kinase activity) causes no detectable suppression of IR resection
2. ATRi has no effect on RPA and therefore resection (ATR is not needed for resection)

Question 13

Figure 7A/B/C

Answer 13

1. A/C: When ATM is inhibited, there is an increase in resection
   1a. ATM is needed to reduce resection

Question 14

Figure 7D

Answer 14

1. DNA-PKcs deficient cells (M059J) exposed to 10 Gy in S phase show in G2 phase enhanced resection that persists for longer time
2. Therefore DNA-PKcs is needed to supress resection

Question 15

Figure 7E

Answer 15

1. DNA PKcs deficient cells
   1a. ATM and DNA-PKcs need to work together for resection