Lecture 15 - CDK mediated regultaion of the G1/S transition

Question 1

What are the four stages of the cell cycle and what occours at each stage?

Answer 1

The Cell Cycle
G1 - Cell responds to growth factors and levels of nutrition
S - DNA Duplication
G2 - Ensure DNA is fully copied
M - Mitosis

Question 2

How does CDK regulate cell cycle transitions?

Answer 2

Each phase of the cell cycle is regulated by a different cyclin. Cyclins accumulate in response to growth factors and transcriptional activation. Cyclin levels oscillate and are degraded after completion of their function. Degradation of the cyclins prevents cell cycle stages reoccurring ensuring forward progression of the cell cycle.

Question 3

How are CDKs regulated

Answer 3

CDKs are regulated at Multiple Levels (G1-S)
Positive regulation
* Mitogen activated signalling
○ Cells receive signals from adjacent cells or mitogens
○ Extracellular signals are mediated from membrane associated receptors to the nucleus
○ Activation of MAPK kinase signalling leads to transcriptional activation of Myc.

* Transcriptional regulation of cyclin expression
	○ Myc promotes cyclin D and E2F expression
	○ E2F is the major transcriptional regulator of S-phase genes and cyclin E/Cyclin A
	○ This leads to DNA synthesis

* Binding of cyclin subunits
	○ CDKs are catalytically inactive in the absence of their cognate cyclin binding partner.
* Activation by phosphorylation of CDK by CDK activating Kinase
	○ CDK when not bound to Cyclin has a loop known as the T-loop which covers its active site. When the cyclin binds the loop extends and is able to be phosphorylated on T160 by the CDK activating kinase (CAK) increasing catalytic efficiency

* Removal of inhibitory phosphorylation sites

Negative regulation
* CDK inhibitor proteins - Bind to active sites and prevent phosphorylation of substrates
○ CDKI protein binds to SPG to prevent S-phase entry
○ The CDK inhibitor protein binds to the cyclin and CDK subunits
○ This ensures that the cyclin-CDK complex is inactive by preventing substrate binding and phosphorylation
○ Transition into S-phase requires degradation of CDK inhibitor
* Inhibitory phosphorylation (wee1)
* Degradation of cyclin subunits by ubiquitin mediated proteasomal regulation

Question 4

Describe how CDKs drive cell cycle progression?

Answer 4

Cyclin levels accumulate then rapidly drop. Cyclins are actively degraded by the proteasome once their function is complete. Sequential cyclin expression provides an increasing CDK activity profile through the cell cycle. This increase in CDK activity, temporally regulates key cell cycle transitions. DNA replication is controlled by distinct CDK activity thresholds.

Question 5

How does CDK activity regulate DNA replication?

Answer 5

CDK activity regulates DNA replication at multiple levels
* DNA replication must occur only once per cell cycle
* This is achieved by separation of replication licensing and DNA synthesis and is regulated by oscillating CDK activity,
* Low CDK activity is required to load DNA factors at origins (DNA replication licencing)
* Intermediate CDK activity inhibits licensing at the G1/S transition and initiates DNA replication
* High CDK activity prevents DNA replication for the remainer of the cell cycle

Regulation of replication by oscillating CDK activity
* Initiation of DNA replication occurs once each cell cycle
* DNA replication must go to completion
* Cells utilize multiple replication origins to ensure timely completion of DNA replication
* The DNA replication origin is recognized by a protein complex known as the origin recognition complex (ORC) that associates with DNA mitosis
* High CDK activity prevents DNA replication - loading of accessory factors only happens at low CDK activity

Pre replication complex assembly
ORC when bound to the origin then interacts with Cdc6 and Cdt1 enabling the recruitment of MCM2-7 helicase. This process is called DNA replication licensing. DNA replication licencing can only occur at low CDK activity i.e. G1 phase. High CDK activity promotes phosphorylation of ORC, Cdc6 and Cdt1. This prevents replication licensing. Phosphorylated Cdt1 is destroyed by the proteasome.

Helicase activation
As CDK activity increases in late G1 phase, the licensed DNA replication origins become activated by recruitment of additional factors that aid MCM loading and activation. Two MCM complexes are recruited to each origin and are able to unwind the helix in opposite directions- therefore replication once initiated is bi-directional from the point of initiation.
MCM is phosphorylated by CDK and DDK to activate its helicase activity - opening dsDNA

Replisome assembly
Recruitment of polymerases and accessory proteins competes replication machinery - replisome