III. Signal transduction & cell cycle | 51. Molecular sensors detecting DNA damage & completion of DNA replication during the cell cycle

Question 1

I. Basics
1. What are the causes of DNA damage?

Answer 1

DNA damage has one of the most important influences on the cell cycle, which can occur as a result of spontaneous chemical reactions in DNA: errors in DNA replication, exposure to radiation or certain chemicals.

Question 2

I. Basics
2. What happen if there are errors in cell cycle?

Answer 2

• It is essential that duplication of genetic material is complete and accurate.
• If cells enter mitosis when DNA is incompletely replicated or damaged, genetic changes will occur.
• These changes can lead to cellular death or, in many cases, to genetic alterations that result in uncontrolled cell growth and division
  => eventually, cancer.

Question 3

I. Basics
3. What is the concept if DNA damage occurs?

Answer 3

If DNA damage occurs
=> cell-cycle arrest immediately
=> try to repair the damage
=> if unsuccessful, the cell must die

Question 4

II. ATR/CHK1 and ATM/CHK2 kinases
1. DNA damage initiates a signaling pathway by activating ___

Answer 4

protein kinases, ATR and ATM

Question 5

II. ATR/CHK1 and ATM/CHK2 kinases
1. DNA damage initiates a signaling pathway by activating ___

Answer 5

protein kinases, ATR and ATM
=> They will associate with the site of damage and phosphorylate various target proteins

Question 6

II. ATR/CHK1 and ATM/CHK2 kinases
2. What is the role of ATR and ATM?

Answer 6

DNA damage initiates a signaling pathway by activating protein kinases, ATR and ATM.
- They will associate with the site of damage and phosphorylate various target proteins

Question 7

II. ATR/CHK1 and ATM/CHK2 kinases
3. What is the mechanism of ATR and ATM kinases?

Answer 7

DNA damage initiates a signaling pathway by activating protein kinases, ATR and ATM. They will associate with the site of damage and phosphorylate various target proteins:
- ATR is activated by a single stranded DNA, and then activates Chk1 (checkpoint kinase 1)
- ATM is activated by double stranded DNA breaks, and then activates Chk2 (checkpoint kinase 2)
- Chk1 and Chk2 will phosphorylate and inhibit Cdc25, preventing it from dephosphorylating and activating Cdk -> Cdc25 will be degraded and no Cdk is activated until replication has been completed and all DNA damages have been repaired
- Chk1+2 will also phosphorylate and activate Wee1,
which in turn will phosphorylate and inactive Cdks -> the Cdks will remain inhibited while replication is still in progress or DNA contains damages
=> ATM, ATR, CHK1+2 will then activate p53 by phosphorylation

Question 8

II. ATR/CHK1 and ATM/CHK2 kinases
4. How is ATR activated?

Answer 8

ATR is activated by a single stranded DNA, and then activates Chk1 (checkpoint kinase 1)

Question 9

II. ATR/CHK1 and ATM/CHK2 kinases
5. How is ATM activated?

Answer 9

ATM is activated by double stranded DNA breaks, and then activates Chk2 (checkpoint kinase 2)

Question 10

II. ATR/CHK1 and ATM/CHK2 kinases
6. How is p53 activated?

Answer 10

• Chk1 and Chk2 will phosphorylate and inhibit Cdc25, preventing it from dephosphorylating and activating CdkCdc25 will be degraded and no Cdk is activated until replication has been completed and all DNA damages have been repaired
• Chk1+2 will also phosphorylate and activate Wee1,
  which in turn will phosphorylate and inactive Cdks -> the Cdks will remain inhibited while replication is still in progress or DNA contains damages
  => ATM, ATR, CHK1+2 will then activate p53 by phosphorylation

Question 11

III. p53
1. What is p53?

Answer 11

p53 is a known as tumor suppressor protein, because its normal function is to limit cell proliferation in the case of DNA damage

Question 12

III. p53
2. Explain the function of p53?

Answer 12

p53 is a known as tumor suppressor protein, because its normal function is to limit cell proliferation in the case of DNA damage:
- In undamaged cells, p53 is highly unstable and present at very low concentrations. This is largely because it interacts with another protein, Mdm2, which acts as a ubiquitin ligase that targets p53 for destruction by proteasomes
- Phosphorylation of p53 after DNA damage (mediated by Chk2) reduces its binding to Mdm2
- This decreases p53 degradation, which results in a marked increase in p53 concentration in the cell. In addition, the ability of p53 to stimulate gene transcription will also increase

Question 13

III. p53
3. What is the mechanism of p53?

Answer 13

When p53 is phosphorylated and activated by ATM, ATR, CH1+2, the phosphorylation will lead to several modifications of p53 that will increase its ability to activate transcription of genes that will help the cell to cope with the DNA damages:
- Hinders ubiquitination (so it is available when needed)
- Favors acetylation (to make DNA more accessible for transcription)
- Exposes the nuclear localization signal (so it can enter the nucleus)
- Hides the nuclear export signal

Question 14

III. p53
4 .When p53 is phosphorylated and activated by ATM, ATR, CH1+2, the phosphorylation will lead to several modifications of p53 that will increase its ability to activate transcription of genes that will help the cell to cope with the DNA damages
=> HOW?

Answer 14

• Hinders ubiquitination (so it is available when needed)
• Favors acetylation (to make DNA more accessible for transcription)
• Exposes the nuclear localization signal (so it can enter the nucleus)
• Hides the nuclear export signal

Question 15

III. p53
5. Explain the structure and mechanism of the active form of p53 protein

Answer 15

• The active form of p53 protein is a stable DNA-associated homo-tetramer.
• Its co-activator is p300, a histone acetyltransferase enzyme. p53 will now be able to transcribe genes needed for cell cycle arrest, apoptosis induction and enhanced DNA repair in response to stress (DNA damage)

Question 16

III. p53 - Activation of p53 at G1 and S phases
6. Describe Activation of p53 at G1 and S phases

Answer 16

Activation by stress, DNA damage, oncogene insult, virus infectionATM, ATR, Chk1+2 p53 activation and Cdc25 degradation
- p53 induces p21 (CKI- Cdk inhibitor proteins) that inhibits Cdk (4,6)-cyclin D (G1 Cdk), Cdk (2)-cyclin E (G1/S Cdk) and Cdk (2)-cyclin A (S Cdk = SPF) complexes
- the inhibition of all these Cdk-cyclin complexes results in inhibition of formation of the pre- replication complex, and thereby inhibition of DNA replication
=> cell cycle is arrested in G1, before the DNA replication occurs in the S phase

Question 17

III. p53 - Activation of p53 at G2-M transition
7A. Describe Activation of p53 at G2-M transition

Answer 17

Activation by stress, DNA damage, oncogene insult, virus infection -> ATM, ATR, Chk1+2 -> p53 activation and Cdc25 degradation
- p53 represses Cdk1 and cyclin B
- p53 induces p21 that inhibits the formation of
Cdk (1)-cyclin B complex (MPF) -> inhibiting
M-phase promoting factor (MPF)
- p53 induces Gadd45 -> Cdk (1)-cyclin B
complex separation to Cdk1 and cyclin B
- induction of 14-3-3sigma -> inactivates MPF in the cytosol and translocates it into the
nucleus
=> Mitosis is inhibited (no active MPF) until stress is relieved

Question 18

III. p53 - Activation of p53 at G2-M transition
7B. at G2-M transition, what does p53 repress?

Answer 18

p53 represses Cdk1 and cyclin B

Question 19

III. p53 - Activation of p53 at G2-M transition
7C. At G2-M transition, what does p53 induce?

Answer 19

• p53 induces p21 that inhibits the formation of
  Cdk (1)-cyclin B complex (MPF) -> inhibiting
  M-phase promoting factor (MPF)
• p53 induces Gadd45 -> Cdk (1)-cyclin B
  complex separation to Cdk1 and cyclin B

Question 20

III. p53 - Activation of p53 at G2-M transition
7D. What is the consequence of induction of 14-3-3sigma?

Answer 20

induction of 14-3-3sigma
=> inactivates MPF in the cytosol and translocates it into the nucleus

Question 21

IV. TGFβ
1. What is TGFβ?

Answer 21

TGFβ is an inhibitor of proliferation

Question 22

IV. TGFβ
2. What is the mechanism of TGFβ?

Answer 22

TGFβ will bind to TGFβ receptors which will activate the SMAD pathway, leading to transcription of CKIs (p15, p21, p27) and lowers production of Cdk4 and Cdc25, so that the cell cycle stops in G1 phase