Cell cycle Flashcards
Which CDK binds with Cyclin D and in which phase is it expressed?
CDK4 and CDK6
G1-phase
Which CDK binds with Cyclin E and in which phase is it expressed?
CDK2
G1-phase
Which CDK binds with Cyclin A and in which phase is it expressed??
CDK2
S-phase
Which CDK binds with Cyclin B and in which phase is it expressed?
CDK1 aka CDC2
G2 and M-phase
Why do cells divide? (what are the cons for being bigger but less rather than smaller but more)
> Lower ability to absorb nutrients and remove waste products
Less gas exchange
Cells less likely to respond to extracellular signalling
Describe flow cytometry? (5 marks)
- Labelled cells suspended in fluid and passed through single file
- laser light focussed on cells
- Excites any flourescence detected
- emitted flourescence detected
- Scattered light detected—> measures total cell number.
What is the amount of flouresence directly proportional to in flow cytometry?
Amount of DNA
More DNA = more intercalation = more flourescence
What is Flow cytometry used for? (there are 4 uses)
Cell counting/sorting
Protein expression
Apoptosis
Cell cycle analysis
What are the 3 common methods to study the cell cycle?
- Light/time lapse microscopy—> cellsin mitosis are rounded up
- DNA binding dyes BrdU, incorporate into new DNA formed in S-phase
- Flow cytometry
What is the function of G1?
Increase the size of the cell, duplicate organelles, PREPARE FOR DNA SYNTHESIS
What is the function of E2F?
TF, stimulates expression of genes needed for the cell cycle
What is Rb?
Transcriptional repressor, blocks the function of E2F
What are the 2 ways Rb inhibits E2F and describe each?
Passive Repression—> Rb binds E2F and prevents it activating transcription of genes nedded for S-phase
Active repression—> Rb recruits the Sin3 & HDAC proteins, Blocks transcription by modifying chromatin
What is the function of HDAC?
HDAC= histone DeAcytylases
Deacetylates histones- closes up chromatin and blocks transcription
Stops polymearse to bind to DNA so no transcription occurs.
Describe the Removal of Active Repression. (4)
- Cyclin-D-CDK4 or 6 —>phosphorylates Rb
- Blocks Sin3 binding to Rb
- HDAC—> repression removed=transcription
- expression of subset of genes needed for late G1 and early S-phase
Describe the Removal of Passive Repression (3)
- Cyclin E-CDK2—> 2nd Rb phosphorylation
- Blocks Rb binding E2F—> removes passive repression of E2F
- Expression of genes needed to complete S-phase:histones, cyclin B, nucleotide synthesis for new DNA
What is the G1 restriction Checkpoint?
Point of no return, checks DNA integrity
What has to happen for the cell to go through G1 checkpoint?
Cells must inactivate: Rb
Rb —> Transcriptional repressor blocks the function of E2F
E2F —> TF, stimulates expression of genes needed for the cell cycle
What cyclins and CDK are in the G1 phase?
Cyclin D + CDK4/6
Cyclin E + CDK2
What cyclins and CDK are in the S?
Cyclin A + CDK2
What cyclins and CDK are in the G2 + M?
Cyclin B + CDK1
What do CKIs do?
Inhibit cell cycle progression
What is active repression by Sin3-HDAC
DNA —> tightly packed around histones
Transcription—> DNA opened up allows polymerase access
Requires histone acetylation
HDACs—> Histone DeAcetylases
De-acetylates histones= closes up chromatin and blocks transcription
What is Ori?
Region of DNA where replication is initiated
Eukaryotes: have multiple Ori’s
Prokaryotes only 1 Ori cause it has a circular chromosome
How is DNA replication controlled?
THERE ARE POINTS FOR EACH STAGE (G1/S)
G1: ORC vinds to Ori
After initiation of cell cycle: E2F partially activated->active repression by Rb
Allows expression of E2F target gene CDC6 which binds to ORC
CDC6, ORC & CDT1 recruit inactive 2x MCM helicase
Binding of MCM helicase to DNA forms the pre-replication complex
At G1/S transition: Cyclin C-CDK2 phosph thepre-replication complex
DNA Helicasesunwind DNA—>inactive DNA polymerase recruited
S: Cyclin A-CDK phosphorylates–> DNA polymerase, CDC6, ORC
Polymearse activated, ORC inactivated->dissociates from DNA, CDC6 targeted for destruction->breaks link between Ori and DNA polymerase
What drug is used to improve elderly health?
Senolytic drugs: kill senescent cells, as SASP is associated with age related disease such as type 2 diabetes, alzheimers etc.
What is a kinetochore?
Large protein structure built at the centromere allows attachment of the spindle microtubules
What is the role of Cyclin B?
B controls G2 and mitosis:
CYTOPLASMIC RETENTION SEQUENCE CRS
NUCLEAR LOCALISATION SEQUENCE NLA
CRS is dominant over the NLS
G2 progresses= increased cyclin B
How is Cyclin B-CDK1 activated?
Inactive: phosphorylated by Wee1, Cyclin B-CDK1 is bound by CRS
CRS phosphorylation by PLK-1 and ERK blocks cytoplasmic retention
Cyclin B-CDK1 enters nucleus remains inactive
PLK-1 phosphorylates and activates CDC25
CDC25 removes CDK1 inhibition
Active cyclin B-CDK1 phosphorylation and furtheractivates CDC25
Positive feedback on cyclin-B-CDK1
Cyclin B-CDK1 can now REGULATE PROGRESSION THROUGH MITOSIS
What is Cohesin?
Forms a ring like structure around the sister chromatid
What is condensin?
Restructures and condenses the DNA
What does Cyclin B-CDK1 do in late G2 and prophase?
Condensin need to be activated
SL1 needs to be inactivated
Cyclin B-CDK1 phosphorylates both. condensin activates, SL1 deactivates
Promotes chromosomal condensation
What does Cyclin B-CDK1 phosphorylate in prometaphase?
Lamin- causes nuclear membrane to breakdown
What is Upiquitin and what are the 3 classes?
Protein degradation
3 classes:
E1 activates ubiquitin
E2 receives Ubq from E1 and prepares it for ligations
E3 ligated Ubq to target proteins
Cyclin B-CDK1 function in metaphase?
Phosphorylates APC and Cdh1 swapped for CDC20
Phosphorylates Seperase and securin binding
What happens in anaphase?
APC-CDC20 ubiquinates securin releasing seperase
Seperase ubiquitinates cohesin (sister chromatids seperate)
Seperase ubiquitinates Fear and MEN- activates CDC14b
APC-CDC20 ubiquitinates Cyclin-B
What happens in Telophase/cytokinesis?
CDC14b is the main factor!!
Dephosphorylates Cdh1 and APC –> swap of CDC20 for Cdh1, so APC-Cdh1 and CDC20 alone?!—> Cyclins Ubiquitinated and destroyed
CDC14b Dephosphorylates myosin–> contractile ring seperates 2 daughter cells
CDC14b dephosphorylated Rb->represses E2F
CDC14b dephosphorylates SL1 so transcription restarts
CDC14b dephosphorylates lamin–> nuclear mem reassembly—> CDC14b –> sequestered in newly forming nucleolus
What happens in the resriction checkpoint(G1)?
HOW IS THE CELL CYCLE ARRESTED
WHAT HAPPENS WHEN THE DNA IS REPAIRED
ATM detects DNA damage causes ATM to become phosphorylated and seperate (they are in pairs).
ATM phosphorylates ChK2–>INDUCE AND STABALISE p53 and inhibit MdM2 is a ubiquitin ligase which targets p53
p53 levels increase—> p21 induced which is a cyclin dependant kinase inhibitor—>Cyclin E-CDK2 inhibited preventing full activation of E2F and preRC
CAUSING THE CELL CYCLE TO ARREST
DNA repaired ChK2 is inactivated so MdM2 is no longer repressed so p53 is ubiquinated–> decrease of p53 and p21 so cyclin E-CDK2 in repressed so entry to S-phase
What happens in the G2/M checkpoint?
DNA damage activates ATR which phosphorylates ChK1 which inhibit CDC25 and activates Wee1
Wee 1 inhibits Cyclin B-CDK1
CDC25 activates Cyclin B-CDK1
CELL CYCLE ARRESTS
IF mutation on wee1 then cell cycle is unregulated: cancer etc.
What happens in the metaphase checkpoint?
Negatively regulates APC-CDC20—> Cyclin B-CDK1 phos Cdh1 and APC
APC dissociates from Cdh1 and binds CDC20
APC-CDC20 ubiquitinates securin
MCC bind and inhibit APC-CDC20
MAD proteins sense if spindle fibre is not correctly positioned etc.
When the sister chromatids are correctly loaded onto the mitotic spindle
MAD and BUB will dissociate from APC-CDC20 so they can ubiquinate Securin
What is the intrinsic apoptosis pathway?
Bcl regulate VDAC and ANT channels
anti apoptotic: Prevent pore opening and cytochrome c leaving the mitochondria
Pro apoptotic: keep pores open and allow cytochrome c to leave the mitochondria
BAX allows cytochrome c to leave throught the VDAC (opening it)
Bcl2 stops VDAC to open for cytochrome c
In high levels BAX can let cytochrome c to leave on it own without VDAC
What happens once cytochrome c is in the cytoplasm?
Cytochrome c and APAF bind together
This causes a cleavage in Caspase 9 to activate it
This activates a cascade to Caspase 3/or 6
caspase 3 can induce apoptosis or caspase 7 and then apoptosis
Caspase 6 only induces apoptosis
