5 - Replication

Question 1

How does the balance between death and survival signalling impact birth and death rate and how is this altered in cancerous cells?

Answer 1

In normal cells, death and survival signalling is in balance, which gives equal birth and death rates. In cancerous cells oncogenes are converted to proto oncogenes, so survival signalling is promoted, and TSGs are inhibited, so death signalling is inhibited.

Question 2

Control over cell proliferation occurs at?

Answer 2

The restriction checkpoint

Question 3

Eukaryotic genes possess many origins of replication, which recruit what and are part of what?

Answer 3

They recruit ORCs, origin recognition complexes, and are part of the PRC; pre replicative complex

Question 4

What must be present for replication?

Answer 4

The pre-replicative complex, Cdk2

Question 5

If Cdk2 action is not present, the DNA..?

Answer 5

Binds the pre initiation complex

Question 6

What is the different between pre and post replicated DNA and what is the role of this?

Answer 6

After replication the pre replication complexis no longer present. If this DNA is transfected into a G1 cell, no replication occurs.

Question 7

Describe Cdk2 activity throughout interphase? How does this impact the PRC?

Answer 7

Cdk2 rises in concentration from zero at G1 stage. The pre-replicative complex only forms in G1, therefore, as it can’t form in the presence of Cdk

Question 8

What does Cdk2 phosphorylate?

Answer 8

ORC and Cdc6

Question 9

What is Cdc6 in the pre replicative complex?

Answer 9

Cdc6 is the initiator part of the PRC, and must be phoshorylated and targeted for degradation to stop replication from starting again.

Question 10

Describe the 3 ways in which re-replication is prevented before mitosis?

Answer 10

1) Generation of the PRC can only occur in the absence of Cdk2; 2) Cdk2 must be present for firing at replication origins; 3) In G2, high Cdk2 prevents any PRCs from forming

Question 11

What is Cyclin E expression controlled by?

Answer 11

Rb

Question 12

Retinoblastoma protein is a substrate of?

Answer 12

Cdks4/6 (+Cyclin D)

Question 13

At what stage of the cell cycle is reetinoblastoma protein regulated?

Answer 13

G1/S transition

Question 14

Describe the impact of phosphorylation on Rb

Answer 14

Hypophosphorylation gives inhibition of the E2F family of transcription factors by recruiting HDAC. Phosphorylated Rb instead recruits HA to E2F transcription factors and stimulates transcription.

Question 15

Describe how Cdk2 activity is impacted by Cdk4/6?

Answer 15

Cdk4/6 regulate the phosphorylation state of Rb, thus determining whether it is inhibitory or stimulatory, and whether the cell cycle will continue. Growth signals stimulate Cdk4/6 to phosphorylate and activate Rb, while anti-growth signals inhibit Cdk 4/6 and therefore Rb is hypophosphorylated and inhibitory on Cyclin E expression

Question 16

Cdk2 activity is also regulated by?

Answer 16

The CKIs p27 & p21, which inhibit Cdk2

Question 17

p27 is controlled by?

Answer 17

Growth signalling; anti-growth signals switch it on

Question 18

p21 is stimulated by?

Answer 18

DNA damage & cell stress via p53

Question 19

Describe the variety of p21/27 targets?

Answer 19

Both target and inhibit Cdk2/Cyclin A/E by interacting with both and distorting the catalytic site. Both also target Cdk4/6, Cyclin D to promote assembly of the complex without inhibition.

Question 20

p27 levels are controlled by?

Answer 20

SCF ubiquitin ligase

Question 21

SCF ubiquitin ligase is degraded in G1 by?

Answer 21

APC/C ubiquitin ligase

Question 22

Describe the specificity and variety of the INK4 family of CKIs

Answer 22

The INK4 family of CKIs consist of p15, p16, p18 and p19. These inhibit Cdk4/6 but not Cdk 2. This is achived by preventing cyclins from binding.

Question 23

Inherited Rb mutations lead to?

Answer 23

Retinoblastoma

Question 24

Inherited p16 mutations lead to?

Answer 24

Familial melanomas

Question 25

Many tumours have aquired mutations in?

Answer 25

p16 and Rb, and also p27 expression is often reduced

Question 26

How does epigenetic silencing occur?

Answer 26

Promoter methylation

Question 27

What is commonly overexpressed in cancers, such as breast cancer?

Answer 27

Cyclin D

Question 28

Medications which target the restriction point checkpoint target where?

Answer 28

They target in the upstream signalling events from the HER2 receptor tyrosine kinase (MAPK, Akt)

Question 29

Why not target Cdks?

Answer 29

The Cdks have function outside of regulation of the cell cycle which could give a large side effect profile. As well as this, Cdk2 -/- mice show viability, suggesting the Cdk2 activity is replaced by Cdk1 in its absense.