Lecture 2 - G1 Control and Restriction Point

Question 1

What is confluence?

Answer 1

When the amount of cells is kept constant by equaling cell division and cell death

Question 2

What happens in normal contact inhibition in a petri dish?

Answer 2

Cells divide until there is no space in the monolayer and then reach confluence

Question 3

What do cancer cells do to do with contact inhibition in a petri dish?

Answer 3

Do not stop dividing and go above the monolayer

Question 4

Where is the restriction point?

Answer 4

Just before S phase

Question 5

What is the restriction point also called?

Answer 5

Rb switch

Question 6

What are cyclin dependent kinases

Answer 6

Heterodimeric protein complex of cyclin and cyclin-dependent protein kinase (Cdk).
Kinases that phosphorylate a selected set of substrate proteins

Question 7

How are cyclin dependent kinases specific?

Answer 7

• Sequence recognition
• Temporal availability (synthesised and destroyed in a cyclic way)
• Spatial location (in the nucleus)

Question 8

Which cyclins are involved in which parts of the cell cycle?

Answer 8

M - cyclin B
G1 - cyclin D
R - cyclin E
S - cyclin A

Question 9

Cyclin levels vary, what happens to Cdk levels?

Answer 9

Kept fairly constant

Question 10

What does CDK1 (CDC2) complex with?

Answer 10

Cyclins A and B

Question 11

What does cyclin D complex with?

Answer 11

CDK4/6

Question 12

What does CDK2 complex with?

Answer 12

Cyclins E and A

Question 13

What happens when you knock out a CDK?

Answer 13

Another CDK will take over

Question 14

What happens when cyclin binds to CDK?

Answer 14

Cyclin pulls the activation loop away from the active site of CDK and exposes the ATP

Question 15

What is CAK and what does it do?

Answer 15

CDK activating kinase.

Required for full activation of CDK

Question 16

How do some CDKi (CDK inhibitors) work?

Answer 16

Block ATP binding at the active site

Question 17

What CDKi’s can inhibit E,A and B?

Answer 17

p57
p27
p21

Question 18

What CDKi’s are needed to inhibit D in G1 phase and what is the family called?

Answer 18

INK4 family
p16
p15
p18
p19

Question 19

How is expression of the INK4 family usually regulated and give an example

Answer 19

Growth factor signalling

Antigrowth signals instigated by TGF B up-regulate p15 resulting in inhibition of cyclin D/Cdk4

Question 20

3 ways pathways can feed into and regulate cyclin D

Answer 20

Activity (CDKi)
Complex formation
Accumulation

Question 21

What activates the expression of p15 and p21?

Answer 21

Dimerisation of Smad3 and Smad4 after 3 is phosphorylated by TGF B receptor kinase

Question 22

What else does SMAD3 do when phosphorylated?

Answer 22

Dimerises with E2F and inhibits Myc - an oncogene

Question 23

What does Myc do when activated?

Answer 23

Inhibits p15 and p21

Question 24

How many cancers is Rb disrupted in?

Answer 24

Probably all

Question 25

How have children with retinoblastoma usually got it?

Answer 25

Usually have inherited one faulty copy and random loss of the second leads to tumour formation (recessive)

Question 26

What happens to the Rb protein as it goes through the restriction point?

Answer 26

Hypophosphorylated (1) pre restriction point

Hyperphosphorylated post restriction point

Question 27

How does the hyperphosphorylation of Rb activate txn?

Answer 27

Removes it from E2F (family of transcription factors)

Question 28

What does the activation of Rb lead to?

Answer 28

Txn of genes required for next cell cycle phase including DNA replication factors

Question 29

What regulates the hypophosphorylation of Rb?

Answer 29

Cyclin D-Cdk4/6

Question 30

What regulates the hyperphosphorylation of Rb?

Answer 30

Cyclin E-Cdk2

Question 31

Why is Rb hypophosphorylated first and what does this mean?

Answer 31

Allows slight txn which includes cyclin E.

Cyclin E is under the control of E2F so unrestricting it leads to massive txn (positive feedback loop)

Question 32

What does E-Cdk2 also phosphorylate and what does this mean?

Answer 32

CDKi p27

Passage through R is irreversible

Question 33

How can cancer mutate to stop the effect of p27?

Answer 33

In some breast cancers it may not become localised to the nucleus effectively

Question 34

Give an example of how DNA tumour viruses render Rb ineffective?

Answer 34

HPV encodes a protein E7 which sits in the pocket of Rb

Question 35

What does Cdk2 activity have an effect on in yeast?

Answer 35

Low Cdk2 activity - supports replication complex assembly in G1
Rising Cdk2 activity - prevents further assembly (to stop multiple repetitions) AND activates complexes

Question 36

How do mammalian cells ensure DNA is only replicated once?

Answer 36

Cyclin E supports replication complex assembly

Cyclin A activates replication complexes and inactivates assembly proteins

Question 37

Describe a pre-replication complex

Answer 37

Origin recognition complex (ORC) binds origin.
Cdc6 cooperates with others to load helicase (Mcm complex)
Presence of helicase defines activatable replication origin

Question 38

What does cyclin A-Cdk2 do in late G1 and early S phase?

Answer 38

Late G1 - Phosphorylates and inactivates Cdc6 and Mcm so they cannot be reused
Early S - phosphorylates DNA polymerases and promotes assembly

Question 39

What is the nuclear matrix?

Answer 39

The structural framework the protein complexes are mounted on

Question 40

What is Ciz1?

Answer 40

Protein that attaches DNA replication complexes to the nuclear matrix

Question 41

What binds to the Ciz1 protein?

Answer 41

Cyclin A and E

Question 42

What happens on Ciz1 when cyclin A levels increase and why?

Answer 42

Cyclin A pushes off E - this coordinates them to function in the right order