Cell Cycle and its Control

Question 1

Internal controls

Answer 1

Monitors progression through cell cycle so that each step happens in succession and delays later events until previous ones are completed

Question 2

External controls

Answer 2

Before beginning cell cycle, cell will sample environment and either proceed with cell division when more cells are needed or block cell division when no cells are needed

Question 3

Two phases of M-phase in cell cycle

Answer 3

1. Mitosis- nuclear division
2. Cytokinesis- cytoplasmic division

Question 4

Why are G1 and G2 needed?

Answer 4

1. To maintain cell mass 2. To monitor internal and external environment

Question 5

Important features of Cell Cycle

Answer 5

1. A clock: turns on specific events at a specific time
2. Initiates events in correct order
3. Mechanism to ensure event is triggered only once per cell cycle

Question 6

Four classes of Cdks

Answer 6

• G1 CDK – promotes passage of cell through restriction point
• G1/S CDK- commits cell to replication
• S Cdk- initiates replication
• M Cdk- promotes mitosis

Question 7

Regulation of replication by S-Cdk

Answer 7

1. In early G1→ ORC binds to origin of replication
2. ORC recruits Cdc6
3. Cdc6 binds and recruits MCM helicases
4. MCM helicases bind to ORC and Cdc6 at the origin
5. The pre-replication complex is assembled
   - Pre-RC is made of

*ORC

*Cdc6

*MCM helicases

6. S-Cdk is activated by S-cyclin
7. S-Cdk phosphorylates Cdc6 thereby causing its release and degradation from the pre-RC
8. MCM helicases are now free to unwind DNA
9. S-Cdk phosphorylates pre-RC complex allowing DNA polymerase to replicate
10. S-Cdk phosphorylates MCM helicases after replication has occurred causing their dissociatioin and degradation
11. Pre-RC falls apart and ORC is also degraded.

Question 8

How is initiation of replication or entry into S-phase controlled?

Answer 8

1. Cdc6 -Cdc6 is usually present at low levels throughout cell cycle.
   - Amount of Cdc6 spikes transiently in G1 -Cdc6 binds

to ORC -This recruits MCM helicases

-The pre-RC is made poised to replicate -S-Cdk

triggers replication and assembles DNA pol and other

replication proteins

2. S-Cyclin
   - The S-Cdk is not active until S-cyclin is activated at the end of G1
   - The switch is the S-Cdk and what turns that switch on is the S-cyclin

*Phosphorylation of Cdc6 and MCM are maintained after S-phase

*M-Cdk also ensures no replication by maintaining phosphorylation of Cdc-6 and MCM helicase

*Cell cycle control system resets itself such that Cdk activity is reduced to 0 at the end of mitosis so newly made Cdc-6 and MCM proteins are not phosphorylated

Question 9

Triggering entry into mitosis

Answer 9

1. M-Cyclin interacts with M-Cdk and partially activates it
   - Partially activates M-Cdk by opening activation site

of M-Cdk a little bit

2. M-Cdk is phosphorylated by CAK (adds one phosphate) in order to make it fully active
3. M-Cdk is held in check by getting additional inhibitory phosphate through phosphorylation by Wee1 kinase
4. M-Cdk is poised to be active when second inhibitory phosphate is taken off by Cdc25 phosphatase.
5. M-Cdk phophorylates proteins responsible for mitosis
   - spindle assembly
   - chromosome condensation
   - breakdown of nuclear envelope

*The switch here is the removal of the second inhibitory phosphate by Cdc25- a phosphatases

Question 10

Positive feedback loop for M-Cdk

Answer 10

1. M-Cdk inhibits Weel upregulating active M-Cdk
   - Cdc25 phosphatase removes inhibitory phosphate

leading to active M-Cdk

• M-Cdk will phosphorylate Wee1 and inhibit it so that

inhibitory phosphate is no longer added to new M

Cdks

2. M-Cdk activates Cdc25 phosphatase increasing rate of production of active M-Cdk
   - M-Cdk will also phosphorylate Cdc25 phosphorylate

and upregulate its activity from being partially active

to completely active

• This increases rate of removal of inhibitory phosphate
• New M-Cdk will never have second inhibitory

phosphate put on because Wee1 is being degraded

by the active M-Cdks made prior to this

Question 11

Negative Feedback Loop for M-Cdk

Answer 11

1. M-Cdk activates ubiquitination of M-cyclin causing degradation of M-cyclin
2. M-cyclin is no longer activating M-Cdk
3. M-Cdk is not activated.

Question 12

Mechanisms to ensure absence of Cdk activity in G1 phase

Answer 12

Mechanisms to ensure an absence of Cdk activity

1. Ubiquitin mediated degradation of Cdks results in no active Cdks in G1 2.
2. Cyclin Kinase Inhibitor (CKI) accumulation
   
   1. Active cyclin-Cdk complex can be bound to p27 which is a cyclin kinase inhibitor (CKI) which then inactivates it.
3. Decreased cyclin transcription
   
   1. No extracellular signal to divide
      
      1. Rb binds to E2F
      2. E2F inhibited
      3. No G1/S cyclins transcribed
      4. S-Cdks inactive
   2. Mitogenic Extracellular signal to divide
      
      1. Mitogenic signal
      2. G1-Cdk accumulates
      3. phosphorylates Rb
      4. Reduces affinity for E2F
      5. E2F transcribes G1/S cyclin genes and E2F transcribes its own genes to up regulate itself
      6. S-cyclins
         
         1. enhance degradation of Ub ligases which degrade CDKs
         2. activate CKI degradationd
         3. activate S-Cdks
      7. S-Cdks phosphorylate Rb upregulates E2F

Question 13

Process of regulation of G1 DNA checkpoint

Answer 13

1. DNA damage detected in G1
2. p53 transcribes CKIs such as p21
3. p21 inactivates G1/S Cdks and S-Cdks
4. Cell does not get past G1 restriction point into S

Question 14

Retinoblastoma

Answer 14

Since Rb protein prevents cell division from occurring, loss of both Rb proteins result in Retinoblastoma

Question 15

Process of regulation of G2 DNA checkpoint

Answer 15

1. DNA damage detected in G2
2. blockage of Cdc25 activity
3. M-Cdk remains inactivated
4. Cell does not get past G2 restriction point into