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What are the 4 stages of the cell cycle?
G1, S, G2, M
At what stage do fully differentiated cells stay arrested?
Where are the checkpoints?
G1/S and G2/M and in the middle of Mitosis
Cells increase in size in the ___ phase.
DNA replication occurs during the ___ phase.
After DNA is synthesized, growth occurs during the ___ phase.
Cell division occurs during the ___ phase.
What is needed to pass a checkpoint?
What are things that might prevent change to S phase?
- Unhealthy cell - e.g. starving.
- Heavily damaged DNA
What might happen if DNA is heavily damaged?
What else is needed to move to the M phase?
- Replicated DNA
- No more than 2 copies of chromosomes
What else is needed to pass from meta to ana?
Chromosomes must be attached to spindles
What are some methods for studying the cell cycle.
- Temp senstive mutants.
- Biochemical experiments with frogs.
- In vitro studies with tissue culture.
- BrdU pulse labeling.
- Flow cytometry.
Describe BrdU pulse labeling.
- BrdU is a thymidine analog.
- Anti-BrdU Abs are used to stain chromosomal DNA
- Provides estimate of duration of each phase
When is BrdU effective?
S and G2 (when chromsomes are duplicated)
How do you know cells are in M phase?
- Look at copies of chromsomes if they're labelled.
- Look for mitotic spindles.
A flow cytometer shows that most cells are in the ___ phase.
What regulates the cell cycle?
- Available space.
- Organ size.
- Proteins (e.g. cyclins)
What are some variations on the cell cycle?
- M -> M
- S -> M
- G1 -> S -> G2 (some plants and insects)
Experiments with fusion between S & G1 and M and G1 indicated what?
Something in S and M activate G1 (G1 is pushed into either of those phases)
Where does the MPF appear to be?
- In the cytoplasm.
- Found by removing cytoplasm from M cell and placing in G1 cell.
___ experiments with frog embryos show that MPF activity is ___.
What does MPF cause?
- Nuclear envelope breakdown.
- Chromosome condensation.
- Spindle fiber formation.
MPF activity is ___ during mitosis and disappears ___.
- at the end of mitosis
Activity of MPF is correlated with the presence of ___.
a protein called cyclin
MPF is composed of ___.
- p34 - CdK - phosphorylates
- p45 - cyclin - activates CdK
Describe the three classes of cyclin.
- G1/S - progression thru start.
- S - stimulate DNA replication.
- M - part of the MPF.
When are cyclins removed?
At the beginning of mitosis.
What cell cycle component is highly conserved?
CdKs, but not CKIs (inhibitors)
CKIs bind to ___.
both cyclin and CdK
How is a CKI removed?
Ubiquitination + proteosome
How are the inhibitory phosphates removed?
How is cyclin degraded?
Proteolysis by APC/C and cdc20 and ubiquitin
What regulates cyclin/CdK activity?
Wee1 inactivates CdK by ___.
Adding two inhibitory phosphates.
Cdc25 reactivates CdK by ___.
Removing an inhibitory phosphates.
CdK also has ___ which is added in ___ and removed in ___ and is required for activity of CdK.
- an activating phosphate
___, not ___, activates CdK.
- Cyclin concentration
Figure 17-21 - overview of cell-cycle control system.
Favorable extracellular environment ___.
DNA damage ___.
inhibits G1/S-CdK, S-CdK, M-CdK
Unrepicated DNA ___.
Chromosome unattached to spindle ___.
- Activates S-Phase.
- inhibits DNA re-replication by initiating degradation of Cdc6.
- inhibits DNA re-replication.
- activates M-Phase.
allows passage thru mid-mitotic checkpoint.
Describe cell division activation via mitogen to G1-CdK in animal cells.
- Mitogen binds to mitogen receptor which activates Ras.
- Ras activates MAP kinase pathway.
- Myc is produced which leads to expression of cyclin genes including G1-CdK.
Describe cell division activation from G1-CdK in animal cells.
- G1-CdK inactivates Rb which activates E2F
- E2F leads to S-phase gene transcription including G1/S-cyclin and S-cyclin.
- G1/S-cyclin and S-cyclin lead to active S-CdK which leads to DNA synthesis.
In cell division activation for animal cells, what provides positive feedback?
- E2F provides positive feedback for itself.
- G1/S-CdK and S-CdK further phosphorylate Rb providing pos feedback for E2F.
Rb protein ___ is required for ___.
- cells to enter the S-phase
- Tumor suppressor protein.
- Trans-acting repressor that inhibits transcription of genes for S-phase.
- Becomes deactivated by phosphorylation by G1-CdK.
- Forms heterodimer with E2F protein.
What is Skp2?
What does Rb-E2F do to Skp2?
- Represses transcription.
- Stimulates Skp2 removal at G0 resulting in no S-phase.
In mid-late G1-phase, what happens with Rb and Skp2?
- Rb is phosphorylated.
- Skp2 transcription increases.
- Skp2 proteolysis decreases, resulting in cell division.
In cells that will divide, what is present at ori sites?
When S-CdK is activated, what happens at ori sites?
- Formation of pre-initiation complex and initiation.
- Replication forks.
When M-CdK is activated, what is the result?
- Chromosome segregation.
At the end of mitosis, what is activated, inactivated, and assembled?
- APC/C activation.
- CdK inactivation.
- Assembly of new pre-replicative complexes at origins.
What proteins are used in replication?
- 1. Gyrase - unwinds supercoils.
- 2. Helicase - unwinds dsDNA.
- 3. Polymerase - adds nucleotides.
- 4. Primase - adds primers (attachment points).
- 5. Ligase
Figure 17-23 - Control of initiation of DNA replication.
Describe the control of the initiation of DNA replication.
- Cdc6 and Cdt1 recruit 6 proteins and form the pre-Replicative Comples (pre-RC).
- S-Cdk stimulates assembly of the pre-initiation complex.
- DNA polymerase et. al are recruited to the origin.
- Mcm protein rings are activated as DNA helicases.
- DNA unwinds.
- Replication begins.
What is geminim?
An APC/C target which inactivates Cdt1.
Describe the processes that prevent re-replication.
- S-Cdk triggers destruction of Cdc6 and inactivation of ORC.
- Cdt1 is inactivated by geminim (an APC/C target).
- Thus, a new pre-RC cannot be formed until end of mitosis.
When is DNA checked for damage and repaired?
- After the pre-replication complex is formed.
- Before S-CdK triggers S-phase.
Polymerase and primase are part of what complex?
The pre-initiation complex.
What do the phosphates do that are attached ORC?
When are the phosphates on the ORC removed?
At the end of M-phase.
What is the first step in replication?
Receiving a signal.
How might replication be prevented?
- Remove receptor of start signal.
- Remove enzymes in signal transduction pathway.
- Remove CdK.
How do differentiation events affect the cell cycle?
- Transcriptional activation of CKIs promotes G0 phase.
- Anaphase promoting complex (APC) and ubiquiting ligase promote G0 arrest.
- Remodeling of the chromatin.
What are examples of differentiation signals that activate CKIs?
Members of the bHLH family including MyoD and Hesl
How does APC promote G0 arrest?
- APC binds to Cdc20 or Cdh1 which activates ubiquiting ligase.
- S cyclins are removed.
- Skp2 is also degraded.
- This results in Rb binding to E2F proteins.
Describe Rb and chromatin remodeling.
- Rb recruits histone deacetylase (HDAC) complex.
- HDAC associates with SWI/SNF ATP-dependent nucleosome remodeling complex.
- Rb binds to enzyme that methylates H3 histones.
- HP1 is recruited and keeps chromatin in repressive state.
What does HDAC do?
Packs DNA together.
What does the SWI/SNF ATP-dependent nucleosome remodeling complex do?
Repackages DNA tightly for activation of replication.
What does HP1 do?
Blocks access to chromatin especially in areas that contain cyclin and promoter genes.
Describe the pathway to inactivate HP1.
There is no such pathway.
What affect does Rb/chromatin remodeling have on differentiated cells?
Those cells can't divide and can't lead to cancer.
What is an easy method to damage DNA?
When DNA is damaged right before cell division, various ___ are recruited that ___.
- protein kinases
- initiate a signaling pathway that causes cell cycle arrest.
What are the first kinases at the site of DNA damage?
ATM or ATR
What is recruited after ATM or ATR?
What do Chk1/2 do?
Phosphorylates p53 preventing Mdm2 binding resulting in CKI p21 production.
What does p21 do?
Binds to G1/S-CdK & S-CdK and inactivates them arresting cell in G1.
When there is no signal for divison, abnormally high levels of Myc cause ___.
activation of Arf.
What does Arf do?
Binds and inhibits Mdm2 thereby increasing P53 levels.
What does stable/active p53 do?
Cell-cycle arrest or apoptosis depending on the cell.