Cell growth + division lecture 4

Question 1

CDC2

Answer 1

• Required for cell cycle transitions
• Wee1 inhibits cyclin B → cyclin B accumulates in late G2 → activates Cdc25 → enters M
• Human G1/S transition is controlled by growth factors, yeast = nutrient

Question 2

Experiment

Identifying mammalian G1-S (cyclin E)

Answer 2

• Took human cDNA library + identified plasmid that complements Cln mutant
• Cyclin E identified
• Binds CDK-subunit + phosph histone
• Looking at northern blot mRNA of cyclin B, appears in regular manner

Question 3

CKI

Answer 3

• Identified w/ Y2H
• p16 binds CDK4/6 + prevents cyclin binding
• p27 blocks ATP binding
• Mitogens inhibit inhibitors p21/-27, release CDK-cyclin D
• p27/p21 cyclinD-CDK4 co-precipitate (needed for formation)
• Nuclear localisation

Question 4

p53

Answer 4

• Proteins of p27 family re-inhibit cyclin-CDK in response to stress
• Stress feeds to p53 TF
• Target of p53 = p21
• ↑ mutation in cancer

Question 5

Mitogen regulation of cyclin D- CDK

Answer 5

• Leads to ↑ responses in Ras/MAPK pathway
• Phosph TF like Ets → binds gene promoter of cyclin D
• cyclin D-p27-CDK4 (inhibit), is phosph by CAK, translocates to nucleus
• PI3K → p27 ↓ by SCF

Question 6

pRB

Answer 6

• pRB-E2F
• pRB-P (by cyclin D-CDK4) x bind E2F → binds gene promoters
• Cyclin E phosph Rb → amplification
• LoF

Question 7

Transcription cell cycle

Answer 7

• Cyclin E phosph cyclin D → destroyed by SCF
• Also phosph MYB, p-MYB → activates MuvB transcription of G2/M genes like cyclin A
• Cyclin A phosph cyclin E → destroyed by SCF
• Cyclin B destroyed cyclin A by APC

Question 8

Resetting the cell cycle APC

Answer 8

• Rb is dephosph on mitotic exit , re-bind E2F
• APC = active, destroys FOXM1
  +ve/-ve feedback

Question 9

DNA replication only 1

Answer 9

• In M, origin of replication bound to DNA + phosph (inactive)
• Geminin sequesters CDT1
• In S, make PIC
• Geminin disappears at start of A
• Cdc6 disappears in late G1
• Narrow window in G1 where can trigger DNA synthesis
• CDT1 destroyed during DNA replication, Gemini uparrow in G2

Question 10

Cell cycle control. of centriole duplication

Answer 10

• Centrioles present in G1
• Centriole duplication regulated by PLK4
• PLK4 activity ↑, autophosph itself
• NEK2