2nd cell cycle checkpoint

Question 1

What are the main roles of the 2nd cell cycle checkpoint

Answer 1

1. Needs to make sure environment is favourable

2. Check all DNA has been replicated before mitosis

Question 2

What role does S-Cdk have

Answer 2

1. S-Cdk activity enhances M-cyclin transcription
2. S-Cdk activates transcription factors (e.g. FoxM1)
3. S-Cdk phosphorylates FoxM1 allowing Plk1 to be able to bind.
4. When Plk1 binds it phosphorylates FoxM1, leading to activation of its transcriptional activity.
5. FoxM1 directly enhances M-cyclin transcription by activating the M-cyclin promoter.
6. FoxM1 is a proto-oncogene.

Question 3

What is M-Cdk activity regulated by

Answer 3

1. M-Cdk activity is regulated by phosphorylation
2. CAK stands for Cdk-activating kinase.
3. The binding of cyclin to Cdk causes the partial activation of the Cdk active site.
4. The phosphorylation of Cdk at an amino acid site located close to the active site results in the full activation; this phosphorylation is initiated by CAK.
5. Further control is exerted by Wee1 kinase and Cdc25 phosphatase.

Question 4

What do Wee1 kinase and Cdc25 do

Answer 4

1. Wee1 causes the phosphorylation of two amino acids above the active site; these phosphate groups are inhibitory and cause the cyclin-Cdk complex to become inactive.
2. Cdc25 on the other hand dephosphorylates these sites allowing the complex to become activate.

Question 5

What happens to M-Cdk at the end of G2

Answer 5

1. At the end of G2 there is an abundance of inactive M-Cdk.
2. Cdc25 is then phosphorylated thereby causing its activation.
3. Activated Cdc25 causes the inactive M-Cdk to become active due to the removal of the inhibitory phosphates.
4. Once M-Cdk is active it phosphorylates and thereby activates further Cdc25, resulting in a positive feedback loop.
5. Active M-Cdk is also able to inhibit Wee1, contributing to this positive feedback loop.

Question 6

What role does subcellular localisation have on M-Cdk

Answer 6

1. M-Cdk is activated first at the centrosomes
2. M-Cdk then moves to the nucleus in late Prophase. 3. Prior to this, M-Cdk is retained in the cytoplasm due to an active nuclear export signal.
3. M-Cdk can only phosphorylate targets at these positions at these times

Question 7

What is the centrosome

Answer 7

1. The organelle located near the nucleus in the cytoplasm that divides and migrates to opposite poles of the cell during mitosis, and is involved in the formation of mitotic spindle, assembly of microtubules, and regulation of cell cycle progression. (definitions taken from biology-online dictionary).

Question 8

What is the SCF complex

Answer 8

1. SCF complex is an E3 ubiquitin ligase
2. Made up of 3 protein subunits
3. F-box protein has specificity for G1 cyclin

Question 9

How do G1/S-cyclin levels fall

Answer 9

1. Ubiquitination
2. Proteins ‘tagged’ with K48-linked polyubiquitin are degraded via the 26S proteasome.
3. The addition of ubiquitin to a protein is a post-translational modification.

Question 10

How is Cdk activity regulated by inhibitor proteins

Answer 10

1. The CKI called p21 binds to both cyclin and Cdk causing a conformational changes in the active site of Cdk rendering it inactive.

Question 11

What does Incomplete DNA replication trigger

Answer 11

1. Incomplete DNA replication triggers the DNA damage response

Question 12

Describe the DNA damage response

Answer 12

1. If stalled replication fork is detected
2. Kinases are recruited to this position- ATE kinases go to position of DNA
3. Chk1 are recruited to the ATR kinases
4. P53 is usually degraded but Chk1 activation causes phosphorylation of p53 causing it become stable and active and dissociate from mdm2
5. P53 can bind to promoter regions of p21 gene causing its transcription
6. P21- inhibitor of m-cdk
7. Chk1 can add additional phosphates to cdc25
8. Stops its expression or activation
9. If cdc25 is not active it can’t remove inhibitory phosphates from mcdk

Question 13

How does DNA damage prevent G1/S-Cdk and S-Cdk activation?

Answer 13

1. p53 is the most frequently mutated protein in cancer
2. DNA damage initiates a signalling pathway by recruiting and activating ATM or ATR which in turn phosphorylate target proteins including Chk2 and Chk1.
3. Chk2 and Chk1 phosphorylate other target proteins leading to cell-cycle arrest.
4. The ATM-Chk2 and ATR-Chk1 pathways are activated by DNA double-strand breaks and single-stranded DNA respectively.
5. The main route of cell-cycle arrest is indirectly through p53 activation.
6. Phosphorylation of p53 causes reduced binding to Mdm2 (a ubiquitin ligase). p53 levels therefore increase within the cell.
7. p53 activates gene transcription of downstream genes, including p21.
8. p21 protein is a Cdk inhibitor; it binds to the G1/S-Cdk and S-Cdk complexes resulting in their inactivation.
9. Without these cyclin/Cdk complexes the cell cannot pass the Start checkpoint and the cell cycle is arrested in G1.
10. If DNA damage is severe and cannot be repaired, the cell will undergo apoptosis.

Question 14

What are the key changes that occur in early mitosis

Answer 14

1. Break down of the nuclear membrane
2. Centrosome separation and maturation
3. Formation and stabilisation of microtubules
4. Chromosome condensation
5. Spindle attachment to sister chromatids

Question 15

What does M-Cdk activate and what does this do

Answer 15

1. M-Cdk activates two protein kinase families
2. M-Cdk, PlKs and Aurora kinases trigger mitosis by the phosphorylation of specific target proteins involved in the assembly and stability of the mitotic spindle and the attachment of the sister chromatids to the spindle.
3. Many of the target proteins which are phosphorylated are yet to be identified.

Question 16

What is the localisation of Plk, Aurora-A and Aurora-B

Answer 16

1. The positioning of mitotic regulators is a key part of their regulation
2. Aurora A- at centrosomes
3. Aurora b- at DNA
4. Plk- centrosomes
5. Plk- at kinetochores