Cell Cycle Flashcards
cell cycle
growth- interphase (longer portion of cycle)
G(0)
G(1) gap- interval between mitosis and dna rep
S phase- dna replication
G2- cell growth and proteins synthesised
division- M phase (mitosis; nuclear division occurs)
prophase
prometaphase
metaphase
anaphase
telophase
time of cell cycle
depends on cell
budding yeast- 90 mins
embryonic cell cycle- 30 mins but has no growth phase
others divide to replace lost cells
process- interphase
G1- cell is diploid (2n= 2 copies of dna), cell is metabolically active + growing
restriction point- (START)
S phase- 4n = 4 copies of dna
cells replicate DNA
occurs once per cell cycle
replication controlled by origin recognition complex (on dna strand) + MCM helicases bind to ORC- displaced from origin after replication and ensure it only occurs once
G2- G2M checkpoint
4 copies of dna (4n)
progression of M phase is triggered by hormonal stimulation
growth factors
if there is enough growth factors, cell can pass through restriction point
without enough GFs, cell will exit cell cycle and enter rest phase called G0
in G1- GF act on cyclin D- dependent on Cdk4/6
s phase- cyclin A paired with Cdk1/2
G2- cyclin B paired with Cdk 1
process- m phase
1hr long
metaphase- anaphase transition (spindle assembly checkpoint)
experiments of cell cycle
phases of interphase identified by dna content
fluorescence intensity using flow cytomotory (measured in fluoresent activated cell sorter- FACS)
cells are incubated with fluorescent dye, binds to dna and identify which stage of cell cycle the cells are in by their dna content
dna content
animal cells in G1- two copies of each chromosome - 2n (diploid)
S phase- in between, where dna is dividing
G2- replication has occurred and DNA content is 4n (tetraploid)
cell cycle experiments
protein kinases trigger major cell cycle transitions
-studies of frog oocytes; arrested in G2 until hormanal stimulation triggers entry into M phase
-genetic analysis of yeasts; found temp sensitive mutants that were defective in cell cycle progression (dna only mutated at ‘wrong’ temps)
-looking at protein synthesis in early sea urchin embryos
frog oocytes
oocytes could be induced to enter M phase by microinjection of cytoplasm from oocytes that had been hormonally stimulated
cytoplasmic factor- maturation promoting factor (now known as cyclin b cdk 1- in G2)
found that MPF is also present in somatic cells - induces entry into M phase
MPF is general regulator of transition from G2 to M (cyclin B cdk1)
genetic analysis of yeast
cdc (cell division cycle) genes required for passage through START and entry into mitosis, encode protein kinases
genetically engineered to have temp sensitive cdc mutant- at permissive temp cell cycle would occur as normal
genetically engineered cell at non permissive temp- would not pass the START point
cdc gene (protein kinase) now has been shown to be cell cycle regulator in all eukaryotes (cdk1)
sea urchin
found two proteins (cyclins) that accumulate throughout interphase but are rapidly degraded at the end of mitosis - suggesting they have a role in including mitosis
found that microinjection of cyclin A was sufficient to trigger G2 to M
cdk1
cyclin dependent kinase 1
adds phosphate group to target group
needs cyclin B to be attached to it
responsible for pushing cells through G2 to M phase
if cyclin b isnt bound to cdk1, cdk1 cannot phosphorylate target proteins and cell cycle doesnt progress
if conditions arent favourable eg temp etc, then cyclin B isnt made and cdk1 cant phosphorylate proteins- cell cycle doesnt progress
overall findings
MPF was purified and shown to be composed of Cdk1 and cyclin B
cyclin B is a regulatory subunit required for catalytic activity of the Cdk1 protein kinase
regulators of cell cycle
regulation of MPF by phosphorylation and dephosphorylation of cdk1
during g2, cyclin B is synthesised and forms complexes with cdk1
cdk1 is phosphorylated and inhibited leading to the accumulation of inactive cdk1/ cyclin b
dephosphorylation activates cdk1 which phosphorylates other proteins that initiate events of M phase
cyclin b is degraded by ubiquitin- mediated proteolysis- leads to inactivation of cdk1, cell can exit mitosis and undergo interphase
ubiquination
post translation modification- adds a tag which signals the protein to be targeted
dna damage checkpoints
throughout interphase
once in each phase (G1, S and G2)
G1- start/ restriction point- once past this point they must commit to going through
S phase-
G2- dna DC= G2M checkpoint
checks if all dna is replicated and if the environment is favourable
skin fibroblasts
arrested in G0 until stimulated by platelet driven growth factor to proliferate and repair wound damage
regulators of cell cycle progression- 4 types
- association of cylin and cdk complex formation
- activating phosphorylation of cdk by cdk activating kinase (CAK)
3- inhibiting phosphorylation by Wee 1
4- binding of inhibitory proteins - cdk inhibitors (CKIs)
intracellular signalling pathways ultimately act to regulate components of the cell cycle machinery
regulators
D type cyclins provide link between growth factor signaling and cell cycle progression
growth factors stimulate cyclin D1 synthesis through Ras pathway (often deregulated in cancer)
growth factor binds to receptor on outside of the cell
(receptor is kinase - phosphorylates ras
ras phosphorylates raf (target protein)
raf phosphorylates mek
mek phosphorylates ERK) which allows it to enter nucleus, bind to dna and promote transcription of target gene (cyclin D1- allows cell to progress)
cyclin d1 is synthesised as long as growth factors are present- allows cell cycle to continue
if growth factors (eg RAS etc) are removed intracellular conc falls rapidly
defects in cyclin D1 regulation contributes to loss of growth regulation characteristics of cancer cells
many human cancers arise as a result of defects in cell cycle regulation
cell cycle + cancer
deregulation of normal growth control can lead to excessive growth of cells- cancer
Rb- tumour suppressor protein/gene (substrate protein for Cyclin D cdk 4,6) - mutated in many human tumours
TSG so inactivation leads to tumour development
normally slow down cell cycle progression
Rb plays a role in coupling cell cycle machinery to expression of genes required for cell cycle progression
Rb action- progression through restriction point
in G0 or early G1, Rb binds to E2F transcription factors
binding suppresses gene expression
Rb is phosphorylated by Cyclin D Cdk 4/6 and dissociates from E2F allowing transcription to occur
progression through restriction point is mediated by activation of cyclin E Cdk2
in G0 and early G1- cyclin E Cdk 2 is inhibited by p27 (inactive)
Cyclin E Cdk 2 can be activated which allows dna transcription to occur
cyclin E synthesised by E2F after Rb is phosphorylated and p27 is inhibited
activation of Cyclin E/ Cdk 2 activated MCM helicase and initiates dna replication (s phase)
cell cycle arrest + dna damage checkpoints
CCA at checkpoints is mediated by protein kinases ATM and ATR- activated in response to DNA damage
dna damage= signalling pathway that leads to cell cycle arrest, dna repair or programmed cell death
ATM- recognises double strand breaks
ATR- recognises single strand breaks or unreplicated dna
they phosphorylate their checkpoint kinases - inhibit cdc25 which normally activates Cdk1/2
cdk 1- arrest in G2
cdk2- arrest in G1 and S phase
p53
arrest at G1 checkpoint is mediated by p53 (TSG)
double strand break (ATM) activates Chk2
phosphorylates p53
binds to dna + increases transcription of specific genes eg p21 which inhibits Cyclin E/Cdk 2- arrest in G1
p53 mutated
often mutated in human cancers
loss of p53 prevents G1 arrest in response to DNA damage so damaged dna is replicated and passed on to daughter cells
tumour mass formed, with damaged dna
