Cell Cycle/Checkpoints

Question 1

Start of Cell Cycle

Answer 1

Rb protein controlled. Early G1 Rb resides in low phosphorylation state associated with TF E2F (inhibited by Rb). Late in G1, cyclin D activates cdk4 and cdk 6 which phosphorylate Rb and releases it from E2F allowing it to transcribe cell cycle protein.

Question 2

S-Phase

Answer 2

1. Origin Recognition Complex (ORC)
2. Cdc6 binds ORC and recruits MCM (pre-replication complex)
3. Cdk-cyclin A and Cdc7-Dbf4 complexes phosphorylate the Cdc6/ORC/MCM complex.
4. Re-initiation is prevented by phosphorylation-dependent poly-Ub of cdc6 by SCF leading to its proteolysis. Phosphorylated form of ORc cannot actively recruit MCM proteins.

Question 3

Mitosis- Entrance

Answer 3

Entrance driven by cyclin B/cdk. Cyclin B accumulates late G2 and kinase activity rises dramatically. Nuclear envelope breakdown, chromosome condensation, spindle assembly.

Question 4

Mitosis- Exit

Answer 4

Anaphase promoting complex (APC). Ub ligase that catalyzes the covalent attachment of Ub peptides to specific target proteins. Then the proteins are degraded by the 26S proteosome. APC catalyzes the Ub of protein scaffolds which leads them to separate. APC also catalyzes the Ub of cyclin B which eliminates the cdk kinase activity and drives cells out of mitosis into G1.

Question 5

G1 arrest induced by DNA damage

Answer 5

Cell will not enter S phase until damage is repaired. p53 critical to this transition.

Question 6

S arrest

Answer 6

Unreplicated DNA. Cell will not leave S phase until all is replicated. Otherwise the chromosomes would tear apart when segregated.

Question 7

G2 arrest

Answer 7

DNA damage.

Question 8

M arrest

Answer 8

Improper spindle assembly. Preventing non-disjunction.

Question 9

DNA Damage Checkpoint

Answer 9

p53 protein. In absence of damage, it is unphosphorylated and binds to Mdm2 which causes the poly-Ub of p53. In damaged cells, kinases ATM/ATR and Chk1/2 are activated and phosphorylate p53. Accumulation in the cells, acts as a TF to activate transcription from the gene encoding the cyclin kinase inhibitor p21, which binds to cyclin E/cdk complex and inhibits its kinase activity which prevents the cell from proceeding.

Question 10

Spindle Checkpoint

Answer 10

Mad2 protein. Cycles on and off kinetochores during mitosis. If no microtubules are bound to a kinetochore, then Bub 1, Bub 3 and Mad 1 complex phosphorylates Mad2, which forms a tetramer and binds Cdc20 subunit of APC and inhibits.

Question 11

cyclin-CDK kinase activity

Answer 11

1. Cyclin association (ie substrate specificity)
2. Phosphorylation (1 spot for kinase activity, the other inhibits)
3. Dephosphorylation (Cdc25 phosphatase removes inhibitory)
4. Cyclin kinase inhibitors (Ink4 and CKI families block kinase)
5. Cyclin Subunit proteolysis (APC/C Ub ligase in M-phase and SCF is Ub ligase for other phases. Cyclin regulation by proteolysis.