Week 5 Holy - Cell Cycle Flashcards
What proteins are responsible for driving the cell cycle?
- Cohesins
- keep replicated chroms together until it is appropriate to separate them
- Condensins
- involved in chromosome condensation
- help pull chroms apart
- Centromeric heterochromatin?
- Spindle architecture?
How is S-phase triggered?
S-Cdk: Cyclin A, CDK 2 or 1
- CDK 2 recruits preinitiation complex proteins to the origin of replication complex (ORC)
- unwinds DNA helix
- loads necessary replication enzymes
- S-Cdk phosphorylates pre-RC proteins, realeasing and destroying them
What are the two primary ways CDK activity is regulated?
- presence of cyclin
- specific pattern of Cdk phosphorylation
- Cdk Inhibitors (CKIs)
What are CkIs?
Cdk Inhibitors
Proteins that bind to the cyclin-Cdk complexes and block the kinase activity of the Cdk.
(also arrest the cell cycle if G1or S if unfavorable conditions)
What is interphase?
- G1, S-phase, and G2
- period when newly divided cells grow to their “adult” size
What is the M-phase?
- Mitosis phase
- accurate segregation of duplicated chromosomes into two complete genomes
- Cytokinesis
- pinching of the cell into two daughter cells
- about 1 hour long
What is G1?
- Phase directly after M-phase
- Decide whether to remain cycling, or stop dividing (enter G0).
- Restriction point (R) or Start = cell is committed to initiating DNA synthesis and entering mitosis.
What is S-phase?
- The period of the cell cycle when DNA synthesis occurs
- chroms replicate
- centrosome replicates
What is G2?
- The period between the completion of DNA synthesis and the onset of M-phase
- chroms begin to condense
- merges into M-phase
When do cohesins release chromatids and allow them to be accurately separated?
When spindle apparatus binds in the M-phase
What do condensins do?
Allow chroms to be separated in a more manageable fashion.
They may form ring-like structures that encircle loops of DNA.
(mechanisms are not well understood)
How is DNA replication triggered in S-phase?
- pre-replicative complex binds when unphosphorylated
- provides substrates for Cdks
- activation of S-Cdk (2or1) → recruits more proteins to form pre-initiation complex
- unwinds DNA → REPLICATION!
How is G1 regulated?
- Growth factors bind to their receptors
- generally tyrosine kinases
- Ligand binding activates Ras g-protein which → activates MAP kinase cascade
- activated MAP kinase is transported into nucleus and activates transcription factors (phosphorylates them)
- produce cyclin D and either Cdk 4 or 6
- phosphorylates Rb → Rb releases active E2F
How are positive feedback loops used cell cycle regulation?
- S-Cdk (cyclin A, Cdk 2 or 1) positively regulates the G1regulation
- helps keep Rb inactivated (by phosphorylation) to provide active E2F
- S-phase gene transcription positively regulates active E2F protein
What are ORCs?
Origin Replication Complexes
- where DNA replication begins
- multiple locations along each chromosome
What are pre-RCs?
- Pre-Replication complex
- complex of initiator proteins that assemble on ORCs
- substrates for Cdks
- only bound when unphosphorylated
- ex. Cdc6, Cdt1, Mcm helicases
Why is overal Cdk activity very low in early G1?
At the end of mitosis and early G1, all Cdk is inactive due to cyclin destruction by anaphase-promoting complex (APC) (allows binding of pre-RC proteins to ORC in early G1).
AND
Because Cdk has to wait to be activated until after all of the building materials for DNA replication are made during in G1.
Approximately how long is the entire cell cycle?
24 hours
Approximately how long is S-phase in a typical mammalian cell?
8-12 hours
Approximately how long is M-phase?
1 hour
What is the purpose of the kinetochore?
Interact with microtubules of the spindle apparatus so that daughter chromatids can be separated.
(also have signaling functions in the metaphase checkpoint)
What is centromeric heterochromatin?
Centromeres of chroms that contain special histone H3 variants that have tightly compacted chromatin (heterochromatin).
During what phase does the centrosome of the cell replicate?
S-phase
How do microtubules move chroms away from the poles in metaphase and separate chroms in anaphase?
Mixture of pushing/pulling forces!
- Microtubule polymerization and depolymerization
- interaction of microtubules with actin/myosin
- activities of dynein (-) and kinesins (+)
What happens during Anaphase A?
chroms move toward poles
shortening of kinetochore microtubules via microtubule depolymerization
What happens during Anaphase B?
Spindle poles move farther apart
(achieved through interaction of interpolar and astral microtubules with motor proteins)
How does the small G-protein Rho help in Cytokinesis?
Organizes actin and myosin into a contractile ring, which constricts, forming a cleavage furrow and pinching the cell into two.
In order to become inactive, M-Cdk (CAK) requires a phosphate group in its inhibitory site, which it receives from what Cdk inhibiting kinase?
Wee1
In order to become active, M-Cdk (CAK) has a phosphate group removed from its inhibitory site by what phosphatase?
Cdc25
What is terminal differentiation?
when cells irreversibly enter G0
(neurons and skeletal muscle cells do not divide)
What is EGF? What does it stimulate?
Epidermal Growth Factor
- molecule secreted to reactivate the cell cycle
- stimulate exit from G0 and a re-entry into G1
What role does E2F have in the transition from G1to S-phase?
E2F turns on genes encoding proteins necessary for S-phase initiation.
Active E2F also promotes transcription of its own gene (positive feedback loop).
When are origins of replication “licensed” (rendered competent for replication?
Early in G1 when pre-RC proteins are unphosphorylated and can bind due to overall low Cdk activity.
What things is M-Cdk responsible for in M-phase?
- chromosome condensation
- nuclear envelope breakdown
- mitotic spindle formation
- fragmentation of the Golgi apparatus and ER
When is M-Cdk activity at its highest?
METAPHASE
corresponds to the peak of M-Cdk activity
How is M-Cdk activity regulated?
- M-cyclin (cyclin B) increased during G2 and M-phase via increased transcription.
- Cyclin B + Cdk 1 = M-Cdk
- Phosphorylated by:
- inhibitory kinase (Wee1) → low activity
- activating kinase (CAK/Cdc25) → increases activity
- Active M-Cdk positively feedbacks to activate more Cdc25
- Active M-Cdk inhibits Wee1 in another positive feedback loop
What are the targets of M-Cdk?
- Condensins
- Centrosomal proteins
- Nuclear pore complexes
- Lamins
- Proteins involved with fragmentation of Golgi and ER
- APC (Anaphase-promoting complex)
Phosphorylation and triggering of the anaphase-promoting complex (APC) by M-Cdk activates its function as a what?
Ubiquitin ligase
-APC targets are ubiquinated and subsequently recognized and destroyed by proteasomes
APC targets include what two things?
- Securins
- protein that inhibits separase (proteolyses cohesins)
- leads to the separation of sister chromatids
- inhibiting an inhibitor
- Cyclin
- destruction of the cyclins brings all Cdk activity to a halt
- reversal of phosphorylation state of Cdk target proteins
What events in M-phase does M-Cdk induce?
First half of Mitosis:
Prophase, Prometaphase, and Metaphase
(also catalyzes its own destruction by activating APC)
What events in M-phase does anaphase-promoting complex (APC) induce?
Anaphase and Telophase
What is the cell’s usual response to DNA damage?
First arrest the cell to allow for DNA repair to occur. Then re-activation of the cycle.
If DNA damage is unable to be repaired, what happens to the cell?
Permanently exit cell cycle to G0(senescence arrest)
OR
Activation of the cell suicide pathway (apoptosis)
What does the cell evaluate at the G1 checkpoint?
Favorable environment to enter S-phase?
DNA damaged after M-phase?
What does the cell evaluate at the G2/M Checkpoint?
Is all DNA replicated?
Is DNA undamaged?
Environment favorable for mitosis?
What does the cell evaluate at the M-phase Checkpoint?
Are all chromosomes attached to the spindle?
When does p53 produce a cellular response to DNA damage?
- START in late G1 (primary response)
- G2/M-phase transition
How does p53 respond to DNA damage?
- serves as a transcription factor to induce the expression of CKIs
- arrest the cell cycle to allow DNA repair to be attempted
- can also trigger cell death if damage is too extensive for repair
- activate gene encoding pro-death protein BAX
- reduces chances of malignant transformation
How is p53 activated in response to DNA damage?
- indirect activation:
- DNA damage → activation of ATM & ATR kinases
- ATM/ATR phosphorylates & activates Checkpoint Kinases 1 & 2
- Chk 1 & 2 phosphorylate p53 → reduced affinity for Mdm2
- Mdm2 usually destroys p53
- inhibition of an inhibitor
- increased p53 → transcription of CKIs (p21)
- CKIs arrest cell cycle → allow DNA repair
- If unsuccessful → p53 promotes cell death
How is the cell cycle arrested during G2?
- ATM/ATR & Chk1/Chk2 kinases are activated
- phosphorylate and inhibit Cdc25 phosphatase → no active M-Cdk
- arrest cell at G2/M transition
What kinase is present in kinetochores and is active in the abscence of attached microtubules?
Bub1
- Active Bub1 inhibits the initiation of cohesin proteolysis, thus inhibiting anaphase onset
Define oncogenes.
Genes which encode products that function in a positive way to promote tumorgenesis.
(mutations that generate oncogenes are gain-of-function)
I.e. gas pedal
Define tumor suppressor.
Genes that encode proteins that are negative regulators of cell growth.
(mutations involving these genes that lead to uncontrolled cell growth are loss-of-function)
i.e. cell cycle “brake pedal”
What are four tumor suppressor genes discussed in class?
- pRB
- p53
- ATM/ATR
- Chk1/Chk2
