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What are the 4 stages of the cell cycle?
G1, S, G2, M
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At what stage do fully differentiated cells stay arrested?
G0
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Where are the checkpoints?
G1/S and G2/M and in the middle of Mitosis
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Cells increase in size in the ___ phase.
G1
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DNA replication occurs during the ___ phase.
S (synthesis)
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After DNA is synthesized, growth occurs during the ___ phase.
G2
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Cell division occurs during the ___ phase.
M
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What is needed to pass a checkpoint?
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What are things that might prevent change to S phase?
- Unhealthy cell - e.g. starving.
- Heavily damaged DNA
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What might happen if DNA is heavily damaged?
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What else is needed to move to the M phase?
- Health
- Replicated DNA
- No more than 2 copies of chromosomes
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What else is needed to pass from meta to ana?
Chromosomes must be attached to spindles
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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.
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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
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When is BrdU effective?
S and G2 (when chromsomes are duplicated)
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How do you know cells are in M phase?
- Look at copies of chromsomes if they're labelled.
- Look for mitotic spindles.
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A flow cytometer shows that most cells are in the ___ phase.
G1
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What regulates the cell cycle?
- Available space.
- Organ size.
- Signals/hormones.
- Proteins (e.g. cyclins)
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What are some variations on the cell cycle?
- M -> M
- S -> M
- G1 -> S -> G2 (some plants and insects)
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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)
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Where does the MPF appear to be?
- In the cytoplasm.
- Found by removing cytoplasm from M cell and placing in G1 cell.
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___ experiments with frog embryos show that MPF activity is ___.
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What does MPF cause?
- Nuclear envelope breakdown.
- Chromosome condensation.
- Spindle fiber formation.
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MPF activity is ___ during mitosis and disappears ___.
- high
- at the end of mitosis
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Activity of MPF is correlated with the presence of ___.
a protein called cyclin
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MPF is composed of ___.
- p34 - CdK - phosphorylates
- p45 - cyclin - activates CdK
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Describe the three classes of cyclin.
- G1/S - progression thru start.
- S - stimulate DNA replication.
- M - part of the MPF.
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When are cyclins removed?
At the beginning of mitosis.
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What cell cycle component is highly conserved?
CdKs, but not CKIs (inhibitors)
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CKIs bind to ___.
both cyclin and CdK
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How is a CKI removed?
Ubiquitination + proteosome
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How are the inhibitory phosphates removed?
Cdc25
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How is cyclin degraded?
Proteolysis by APC/C and cdc20 and ubiquitin
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What regulates cyclin/CdK activity?
phosphorylation/dephosphorylation
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Wee1 inactivates CdK by ___.
Adding two inhibitory phosphates.
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Cdc25 reactivates CdK by ___.
Removing an inhibitory phosphates.
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CdK also has ___ which is added in ___ and removed in ___ and is required for activity of CdK.
- an activating phosphate
- G2
- mitosis
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___, not ___, activates CdK.
- Phosphorylation
- Cyclin concentration
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Memorize:
Figure 17-21 - overview of cell-cycle control system.
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Favorable extracellular environment ___.
activates G1-CdK
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DNA damage ___.
inhibits G1/S-CdK, S-CdK, M-CdK
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Unrepicated DNA ___.
inhibits M-CdK
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Chromosome unattached to spindle ___.
inhibits APC/C
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S-CdK ___.
- Activates S-Phase.
- inhibits DNA re-replication by initiating degradation of Cdc6.
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M-CdK ___.
- inhibits DNA re-replication.
- activates M-Phase.
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APC/C ___.
allows passage thru mid-mitotic checkpoint.
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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.
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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.
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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.
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Rb protein ___ is required for ___.
- inactivation
- cells to enter the S-phase
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Describe Rb.
- 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.
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What is Skp2?
Ubiquitin ligase.
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What does Rb-E2F do to Skp2?
- Represses transcription.
- Stimulates Skp2 removal at G0 resulting in no S-phase.
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In mid-late G1-phase, what happens with Rb and Skp2?
- Rb is phosphorylated.
- Skp2 transcription increases.
- Skp2 proteolysis decreases, resulting in cell division.
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In cells that will divide, what is present at ori sites?
pre-replicative complexes.
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When S-CdK is activated, what happens at ori sites?
- Formation of pre-initiation complex and initiation.
- Replication forks.
- Elongation.
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When M-CdK is activated, what is the result?
- Chromosome segregation.
- Mitosis.
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At the end of mitosis, what is activated, inactivated, and assembled?
- APC/C activation.
- CdK inactivation.
- Assembly of new pre-replicative complexes at origins.
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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
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Memorize:
Figure 17-23 - Control of initiation of DNA replication.
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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.
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What is geminim?
An APC/C target which inactivates Cdt1.
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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.
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When is DNA checked for damage and repaired?
- After the pre-replication complex is formed.
- Before S-CdK triggers S-phase.
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Polymerase and primase are part of what complex?
The pre-initiation complex.
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What do the phosphates do that are attached ORC?
Prevent replication.
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When are the phosphates on the ORC removed?
At the end of M-phase.
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What is the first step in replication?
Receiving a signal.
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How might replication be prevented?
- Remove receptor of start signal.
- Remove enzymes in signal transduction pathway.
- Remove CdK.
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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.
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What are examples of differentiation signals that activate CKIs?
Members of the bHLH family including MyoD and Hesl
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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.
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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.
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What does HDAC do?
Packs DNA together.
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What does the SWI/SNF ATP-dependent nucleosome remodeling complex do?
Repackages DNA tightly for activation of replication.
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What does HP1 do?
Blocks access to chromatin especially in areas that contain cyclin and promoter genes.
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Describe the pathway to inactivate HP1.
There is no such pathway.
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What affect does Rb/chromatin remodeling have on differentiated cells?
Those cells can't divide and can't lead to cancer.
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What is an easy method to damage DNA?
Water
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When DNA is damaged right before cell division, various ___ are recruited that ___.
- protein kinases
- initiate a signaling pathway that causes cell cycle arrest.
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What are the first kinases at the site of DNA damage?
ATM or ATR
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What is recruited after ATM or ATR?
Chk1/Chk2
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What do Chk1/2 do?
Phosphorylates p53 preventing Mdm2 binding resulting in CKI p21 production.
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What does p21 do?
Binds to G1/S-CdK & S-CdK and inactivates them arresting cell in G1.
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When there is no signal for divison, abnormally high levels of Myc cause ___.
activation of Arf.
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What does Arf do?
Binds and inhibits Mdm2 thereby increasing P53 levels.
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What does stable/active p53 do?
Cell-cycle arrest or apoptosis depending on the cell.
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