L2-3- cell cycle Flashcards
what are the main 5 stages of the eukaryotic cell cycle?
Interphase: G1 phase S phase G2 phase Mitosis: M phase cytokinesis
how long does a complete cell cycle last in actively growing mammalian cells?
24 hours
why is yeast a good model for cell cycle control?
small genomes, easy to make mutants, rapid growth - cell cycle can be as short as 2 hours
what genes do mutants have mutations in?
CDC genes - (cell division cycle)
what are the main ‘checkpoints’ in the cell cycle that control progression onto the next stage of the cycle?
- commitment to DNA synthesis - in G1 (restriction point in mammals)
- commitment to enter Mitosis - in G2
- commitment to complete mitosis - in M
how is the cell cycle controlled?
by protein phosphorylation and dephosphorylation
proteins that catalyse reactions in processes of the cell cycle regulated by
what are Cdks?
cyclin-dependent protein kinases
these are the principle enzymes that control the cell cycle, only active in presence of specific proteins called CYCLINS
What trigger each stage of the cell cycle?
specific cyclin-Cdk complexes
how many Cdks do vertebrates have compared to yeast?
4 Cdks, several cyclins, whereas yeast only has 1 Cdk
what are the 4 classes of cyclin?
In most cells: G1-cyclins (in late G1 phase) - control initiation of processes IN ALL CELLS: G1/S-cyclins - initiate S phase S-cyclins - initiate DNA synthesis M-cyclins - initiate mitosis
what is the role of cyclin?
cyclin directs Cdk to phosphorylate specific target proteins
what are 2 main specific target proteins of cyclin-Cdk complexes?
Lamin - protein of nuclear lamella
Condensin complex proteins - required for chromosome condensation
what controls cyclin concentrations?
the concentrations are determined by the balance of:
- cyclin synthesis - cyclins accumulate during cell cycle
- cyclin degradation - by proteolysis
what are proteasomes?
Large complex involved in breakdown of proteins which is ATP dependent with two main parts:
- hollow cylinder in which proteolysis takes place
- cap that binds and unfolds proteins to be destroyed (associated with ATP hydrolysis)
they degrade proteins with small protein UBIQUITIN attached
what is the protein ubiquitin?
attaches to target protein (e.g. cyclins) with degradation signal - added via ubiquitin ligases which add the chains of ubiquitin polypeptides to target protein marking them for destruction
which carboxy-terminal of ubiquitin is attached to the amino group of a lysine side chain on the target protein?
GLYCINE - forms peptide bond
how do ubiquitin ligases identify target proteins?
recognise degradation signals on the target proteins, these signals can be:
- non-specific - proteins that have abnormal structures
- specific - proteins with short lives
are ubiquitin ligases specific or not?
yes - cells contain multiple ubiquitin ligases, different ones recognise different degradation signals
Cdk activity is regulated by…
- concentrations of cyclins
- phosphorylation and dephosphorylation of Cdk itself
- Cdk inhibitor proteins (CKIs) - bind to cyclin-Cdk complexes and inhibit activity
which cyclin-Cdk complexes are involved at these main control points of the cell cycle:
- G1 to S transition?
- entry into mitosis?
- transition to anaphase?
- G1 to S transition - involves G1-Cdk, G1/S-Cdk and S-Cdk
- entry into mitosis - involves M-Cdk
- transition to anaphase - involves M-Cdk
The G1 to S transition
- start of G1 = no cyclins present
- G1-cyclin accumulates in G1 and G1-Cdk activates G1-S-cyclin gene transcription
- G1/S-Cdk increases S-Cdk activity by: activating transcription of S-cyclin genes, inhibiting an S-Cdk ubiquitin ligase (more S-Cdk), and inhibits an S-Cdk inhibitor protein (activating S-Cdk)
- S-Cdk promotes DNA synthesis
what triggers the G1 to S transition?
external signals mechanism, in animals: MITOGENS stimulate cell division, GROWTH FACTORS stimulate increase in mass. One protein can have both activities, e.g. Platelet-derived growth factor (PDGF) - stimulates many cells to divide
Erythropoietin - induces proliferation of red blood cell precursors
what are Mitogens?
External signals that stimulate cell division, they bind to cell surface and indirectly increase cyclin activity
e.g. stimulate transcription of G1-cyclin genes
cause degradation of G1/S-Cdk inhibitor proteins
what is the Retinoblastoma protein (RB1)?
AN INHIBITOR in normal cells: prevents premature cell division by blocking transition to S phase - it inhibits transcription of genes for G1/S-cyclin and S-cyclin by binding to transcription factor E2F
what inactivates RB1 protein by phosphorylating it?
G1-Cdk (inhibits RB1 whch is an inhibitor)
what is Retinoblastoma in children?
inherited eye cancer caused by loss of both copies of RB1 gene, RB1 protein functions in many other cell types.
what happens after G1-Cdk inactivates RB1 protein?
genes coding for G1/S-cyclin and S-cylcin ARE transcribed so G1/S-Cdk and S-Cdk start to accumulate
what happens once G1/S-Cdk and S-Cdk start to accumulate?
they inactivate RB1 protein too
EF2 also stimulates own transcription
what is the key protein controlling response to DNA damage called?
p53 - accumulates when DNA is damaged
normally in very low levels
mutations in this occur in many forms of cancer
what is the normal mechanism for p53 and what happens when DNA is damaged?
p53 is ubiquitinated and destroyed
DNA damage leads to PHOSPHORYLATION of p53 which prevents addition of ubiquitin
What happens when p53 is phosphorylated?
activates transcription of gene for p21 - a Cdk inhibitor protein
what is p21?
a Cdk inhibitor protein, inhibits G1/S-Cdk and S-Cdk allowing time for DNA repair
summary of G1 to S transition
SYNTHESIS of G1-cyclin, G1/S-cyclin, S-cyclin increases
G1/S-Cdk prevents inhibition of S-Cdk and stabilises it
MITOGENS promote synthesis of G1-cyclin and prevent inhibition of G1/S-Cdk
RB1 PROTEIN prevents early increase in G1/S-cyclin, S-cyclin concentrations and their corresponding Cdk activities
DNA damage causes stabilisation of p53 causing p21 to accumulate - inhibiting G1/S-Cdk and S-Cdk
how does entry into mitosis occur?
M-cyclin synthesis increases in G2
M-cyclin binds to Cdk = M-Cdk
M-Cdk phosphorylated by wee1 kinase inhibiting Cdk activity, then reactivated by Cdc25 phosphatase by dephosphorylating it
why is Cdc25 phosphatase activated in late G2 phase?
it is phosphorylated by POLO-LIKE KINASE - activity of polo-like K increases during G2/M phases
what is Polo-like kinase?
polo-like kinases are common in eukaryotes
activity increases during G2/M phases
phosphorylate Cdc25 phosphatase
what are cyclins?
group of proteins that control progression through cell cycle by activating cyclin-dependent kinase enzymes
what happens during inhibitory phosphorylation by Wee1 kinase?
M-Cdk phosphorylated by wee1 kinase inhibiting Cdk activity
late G2 phase: Cdc25 phosphatase is activated by phosphorylation - Cdc25 dephosphorylates M-Cdk activating it
M-Cdk then activates Cdc25 phosphatase and inhibits wee1 kinase = positive feedback
what happens during phosphorylation by Cdk-activating kinase
full activation of cyclin-Cdk requires phosphorylation by Cdk-activating kinase at the active site
what is the purpose of he DNA replication checkpoint?
incomplete replication of DNA damage results in Cdc25 phosphatase not dephosphorylating M-Cdk, therefore not progressing to mitosis
what happens in anaphase?
sister chromatids separate
by separase protease cleaving subunit of cohesin
what holds sister chromatids together prior to anaphase?
cohesin complex
what are the steps in the action of separase?
- before anaphase: securin inbihits separase
- during mitosis: M-Cdk activates APC by promoting binding of Cdc20
- ABC ubiquitinates securin - degraded
what is APC?
Anaphase promoting complex - can be called cyclosome
a ubiquitin ligase
what is the exit mechanism from MITOSIS?
- APC adds ubiquitin to M-cyclin
- M-cyclin is destroyed
- APC inactivated in absence of M-cyclin
