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

depends on cell budding yeast- 90 mins embryonic cell cycle- 30 mins but has no growth phase others divide to replace lost cells

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

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

1hr long metaphase- anaphase transition (spindle assembly checkpoint)

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

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)

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

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)

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)

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

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

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

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

post translation modification- adds a tag which signals the protein to be targeted

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

arrested in G0 until stimulated by platelet driven growth factor to proliferate and repair wound damage

• 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

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

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

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)

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

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

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