Control of eukaryotic cell cycle

Question 1

Explain G2/M transition

Answer 1

Cdc25 vs Wee1
Phosphorylation of cdc2
Cdc2 and cdc13 is the MPF

Question 2

Genetic screens

Answer 2

cdc-ts phenotype
wee phenotype
overexpression screen
allele-specific suppressions (cdc2, cdc13) and double mutants (cdc25/wee1)

Question 3

4 ways of regulation of CDK1 (cdc2)

Answer 3

Phosphorylates wee1/cdc25 (positive feedback)
Phosphorylates and masks NES (by itself and PLK1)
ATR –> CHK1 –> cdc25 (also via 14-3-3), wee1
cdk1 –> Greatwall –> ENSA –> PP2A-B55d

Question 4

APC/C co-activators

Answer 4

cdc20 - degrades destruction box (CycB, Securin)

cdh1 - degrades KEN box and destruction box (PLK1, Aurora, Cdc20)

Question 5

SAC

Answer 5

Kinetochores unattached: Displays MAD1/2 complex, catalyses binding of another MAD2 complex to cdc20, binds to Bub3 and BubR1, which cannot activate APC/C

Attachment allows Cdc20 to associate with APC/C

Question 6

Removal of cohesins

Answer 6

Cohesins distal from centrosomes removed by phosphroylation by PLK-1

SAC fulfilled, APC/C:cdc20 ubiquitinates securin (via destruction box), which releases separase, cleaves cohesin at SCC1 subunit

Scc1 cleavage is necessary (removal of both cleavage sites) and sufficient (replacement with TEV) for chromatid separation - Scc1 mutants have premature separation

Question 7

G1/S/G2

Answer 7

M-phase: APC/C mediates Cdc2/Cdc13 degradation
G1: Rum1 inhibits Cdc2/Cig2 and Cdc2/Cdc13

Late G1: Cig1/cdc2 Puc1/cdc2 inhibits Rum1, allowing Cdc2/Cig2 to accumulate

Start of S-phase: Cig2/Cdc2 accumulates, inhibits APC/C

Cdc2/Cdc13 accumulates but remains inactive by wee1 and Mik1

Question 8

Mik1 and wee1

Answer 8

Cdc2-Y15F enters M phase from any phase in the cell cycle, but wee1 mutants only enter prematurely from G2 phase.

wee1 and mik1 double mutants have same phenotype as cdc2-Y15F mutant

Question 9

Genes that are the minimal cell cycle machinery

Answer 9

Sufficient to drive cell cycle:
Cdc2-Cdc13 fusion gene is expressed under a cdc13 promoter:
if endogenous cdc2, cdc13, cig1, cig2, and puc1 genes are knocked out, cells can still divide

Necessary to drive cell cycle:
o Gatekeeper mutation in cdc2
o ts-mutation in the cdc13 gene

cdc2- cdc13- cells can divide, but arrest in G2 when adding inhibitor or restrictive temperature.
cdc2- cdc13- cig2- have delayed onset of S phase - shows Cig2 drives S phase but is not necessary

Question 10

Show that it’s fluctuations that drive cell cycle

Answer 10

Fluctuations in cdc2/cdc13 drive cell cycle:
o Gatekeeper mutation in cdc2
o ts-mutation in the cdc13 gene

In cdc2- cdc13- cig2- cig1- puc1- cells: low inhibitor arrests in G2, high inhibitor arrests in G1

In cdc2- cdc13- cig2-
Express inhibitor-sensitive cdc13/cdc2 fusion protein with destruction box removed under an inducible promoter.
Cell cycle can be driven by fluctuations of inhibitor concentrations. Low cdc2/13 activity needed to drive G1/S, while high cdc2/13 needed to drive G2/M