L15: Cdk mediated regulation of G1/S transition

Question 1

How do cyclin levels change throughout the cell cycle? - what stimulates their accumulation?

Answer 1

Each cyclin is expressed in a particular phase of the cell cycle. Cyclins accumulate in response to growth factors and transcriptional activation, and are degraded after completion of their function; prevents stage form reoccurring
-> directionality
- DNA replication is controlled by distinct cdk activity thresholds

Question 2

Positive regulation (levels of control of cdks):

Answer 2

Positive regulation (activation):
- Mitogen activated signalling.
- Transcriptional regulation of cyclin expression
- Activation by phosph. of cdk (by CAK)
- Removal of inhibitory phosph. sites (cdc25 - remove phosph. of Tyr14, Thr15)

Question 3

Mitogen dependent signalling

Answer 3

• Cells receive signals from adjacent cells or mitogen
• Extracellular signals are mediated from membrane associated receptors to the nucleus
• Activation of MAPK kinase signalling leads to transcriptional activation of Myc

Question 4

Action of Myc protein

Answer 4

• Monophosphorylates Cdk (many potential sites)
• At a low level, ability of E2F to promote transcription is activated
  -> sufficient to begin production of cyclin E
• Positive feedback loop established
• Hyperphosphorylation of Rb -> weakened affinity for E2F - released, enhances feedback

Question 5

Negative regulation (levels of control of cdks):

Answer 5

• Cdk inhibitor proteins; bind to AS and prevent phosph. of substrates
• Inhibitory phosph. (Wee1)
• Degradation of cyclin subunits by ubiquitin mediated proteosomal regulation

Question 6

CDKI proteins

Answer 6

• Bind to SPF to prevent S-phase entry
• Binds to cyclin and cdk; ensures complex is inactive by preventing substrate binding and phosph.
• Must be degraded if a cell is to enter S-phase

Question 7

Licensing vs replication

Answer 7

• Replication licensing and DNA synthesis are separate and highly regulated by osculating cdk activity
• Low cdk activity required to load DNA factors at origins
• Intermediate cdk activity inhibits licensing at G1/S transition -> replication

Question 8

Pre-Rc assembly/ Licensing process and how it is prevented

Answer 8

• ORC, when bound to origin, interacts w/ Cdc6 and Cdt1 enabling recruitment of MCM2-7 helicase (DNA replication licensing)
• High cdk activity prevents this by phosph. ORC, cdc6, cdt1
• Phosph. cdt1 is destroyed by the proteoasome

Question 9

Discuss regulation activation of replication (wrt irreversibility)

Answer 9

Following formation of PreRC under low [Cdk]…
1. S-phase Cdk, DDK phosph. members of PreRC; they dissociate -> irreversible
2. Additional factors dissociate, MCM helicase activated (by Dbf4 dependent kinase/DDK) and DNA helicase activated, DNA helix unwound at origin
3. DNA pols loaded, replication initiated (replisome formed)