Biochemical Control of the Cell Cycle

BNS230

Lecture programme

• Three lectures

• Aims– Describe the cell cycle– Discuss the importance of the cell cycle– Discuss how the cycle is regulated

S-phase(DNA synthesis

G1 phase

Mphase

G2 phase

G0 state

Cell division

5 hours

12 hours

15 hours

16 hour cell cycle

Cell cycle definition

• A series of distinct biochemical and physiological events occurring during replication of a cell

• Occurs in eukaryotes

• Does not occur in prokaryotes

• Time of cell cycle is variable

Cell cycle timing

• Yeast 120 minutes (rich medium)• Insect embryos 15-30 minutes• Plant and mammals 15-20 hours• Some adults don’t divide

– Terminally differentiated– e.g. Nerve cells, eye lens

• Some quiescent unless activated– Fibroblasts in wound healing

Components of the cell cycle

• M phase– Cell division– Divided into six phases

• Prophase• Prometaphase• Metaphase• Anaphase• Telophase• Cytokinesis

Components of the cell cycle

• G1 phase– Cell checks everything OK for DNA

replication– Accumulates signals that activate

replication– Chloroplast and mitochondria division not

linked to cell cycle

Components of the cell cycle

• S-phase– The chromosomes replicate– Two daughter chromosomes are called

chromatids– Joined at centromere– Number of chromosomes in diploid is four

Components of the cell cycle

• G2-phase– Cell checks everything is OK for cell

division– Accumulates proteins that activate cell

division

Why have a cell cycle?

• Comprises gaps and distinct phases of DNA replication and cell division

• If replicating DNA is forced to condense (as in mitosis) they fragment

• Similarly if replication before mitosis– Unequal genetic seperation

• I.e. Important to keep DNA replication and mitosis separate

Why have a cell cycle?

• Important to have divisions in mitosis• e.g. Important metaphase complete

before anaphase. Why?• If not segregation of chromosomes

before attachment of chromatids to microtubles in opposite poles is possible

• Down syndrome due to extra chromosome 21

Why have a cell cycle?

• Gaps provide cell with chance to assess its status prior to DNA replication or cell division

• During the cell cycle there are several checks to monitor status

• These are called checkpoints

Checkpoints

• Checkpoint if G1 monitors size of cell in budding yeast (Saccharomyces cerevisae)

• At certain size cell becomes committed to DNA replication

• Called start or replication site

Evidence of size checkpoint

• Yeast cells (budding yeast) grown in rich medium

• Switch to minimal medium• Cells recently entering G1 (buds)

delayed in G1 (longer to enter S-phase)• Large cells above threshold size still go

to S-phase at same time as in rich medium

Evidence of size checkpoint

• Yeast in rich medium– 120 minute cell cycle

• Short G1 phase

• Yeast in minimal medium– Eight hour cell cycle primarily because of

long G1 phase

Checkpoints

• Checkpoint 2 in G1 monitors DNA damage

• Evidence?– Expose cells to mutagen or irradiation– Cell cycle arrest in either G1 phase or G2

phase

• The protein p53 involved in cell cycle arrest – Tumour suppresser

Checkpoints

• Checkpoint in S-phase monitors completion of DNA replication– Cell does not enter M-phase until DNA

synthesis is complete

• Checkpoint in G2– DNA breaks cause arrest– Otherwise when chromosomes segregate in

mitosis DNA distal to breaak won’t segregate

Checkpoints

• Checkpoint in mitosis– Senses when mitotic spindles have not formed– Arrests in M-phase– Otherwise unequal segregation of

chromosomes into daughter cells

• Described cell cycle, now I will talk about genes and proteins that control this process

Molecular control of cell cycle

• Two experimental approaches– Biochemical

• Sea urchin fertilised eggs• Rapid• Synchronous division• Analyse proteins at various stages of cycle

– Genetic analysis using• Budding yeast Saccharomyces cerevisae• Fission yeast Schizosaccharomyces pombe

Using genetics to study the cell cycle

• To study the genetic basis of a biological event– Make mutants defective in that event– Determine which genes have been mutated– Understand role of gene (and encoded protein)

in the event– Problem: How do you make mutants that disrupt

the cell cycle– Cells will not replicate

Using genetics to study the cell cycle

• Isolate temperature sensitive mutants that have defect in cell cycle

• At low temperature these mutants progress through cell cycle

• Arrest in cell cycle at elevated temperature

• Mutation causes gene product (protein) to be highly sensitive to temperature

Using genetics to study the cell cycle

• Isolation of genes that regulate the cell cycle

• Step 1: Create strains with mutations in cell cycle genes

Isolating cell cycle mutants

Yeast culture(S. pombe)

Mutagenise and plate out at high and low temperature

37°C30°C

Colonies 4 and 10 are possible cell cycle mutants. Calledcell division cycle (cdc) mutants >70 cdc mutants isolated

Are the temperature sensitive mutants cdc mutants?

Grow colonies at 30°CShift temperature to 37°CLook under a microscope

Colony 4: Too small; enters mitosis tooearly (Wee 1 mutant)

Colony 10: very long stuck in G2 (cdc25 mutant)

Wild type cells

Using genetics to study the cell cycle

• Step 2: Insert plasmids containing fragments of wild type DNA

• Step 3: Look for plasmid that corrects genetic defects

• Step 4: Plasmid contains a cell cycle control gene

What do we do with the mutants?

Use mutants to isolate cdc genes andthen study what the proteins do

WildtypeS. pombe

Extract DNA

Wee1 cdc25

Yeast vector

Cut with restriction enzyme and ligate into vector

Take recombinant vectors and transform into cdc mutants

• Wee mutant with normal gene wee1 gene in plasmid will grow at 37

• cdc25 mutant with normal cdc25 gene in plasmid will grow at 37

• I.e gene in recombinant plasmid is complementing the mutation

Biochemical studies

• 1st evidence proteins regulate cell cycle– Fuse interphase cells (G1, S or G2) withM-

phase cells– Cell membranes breakdown and

chromosomes condense – I.e Mitotic cells produce proteins that cause

mitotic changes in other cells

Microinjection with frog oocyte

• Oocyte stays in G2-phase• Male gets busy and female produces

progesterone• Oocyte enters mitosis• Purify proteins from oocyte cells treated

with progesterone• Inject into G2 arrested cells and see

which protein causes mitosis (1971)

MPF

• Protein identified that causes mitosis

• Called maturation promoting factor

• MPF in all mitotic cells from yeast to humans

• Renamed mitosis-promoting factor

Properties of MPF

• MPF activity changes through the cell cycle

• MPF activity appears at the G2/M interphase• and then rapidly decrease

How does MPF cause mitosis?

• It’s a protein kinase– Phosphorylates proteins

• Phosphorylates proteins involved in mitosis• Phosphorylates histones causing chromatin

condensation• Phosphorylates nuclear membrane proteins

(lamins) causing membrane disruption

Characterisation of MPF• Consists of two subunits; A and B• Subunit A: Protein kinase• Subunit B: Regulatory polypeptide called cyclin

B• Protein kinase present throughout cell cycle• Cyclin B gradually increases during interphase

(G1, S, G2) • Cyclin B falls abruptly in anaphase (mid-

mitosis)

G1 S G2 M

Protein kinase (subunit A)

Cyclin B levels (subunit B)

MPF activity

What does this profile tell you?

MPF not just due to association of subunits A and Bother factors involved

Anaphase

Telephase

Interphase(G1-S-G2)

Prophase

Metaphase

Ubiquitin

Proteosome

Cyclin B (subunit B) Protein kinase (subunit A)

MPF}

Cyclin B

• How do Cyclin B levels decrease abruptly• Proteolytic degradation• Degraded in a protease complex present in

eukaryotic cells called “The Proteosome”• Specific proteins degraded by complex

when tagged by a small peptide called ubiquitin

Cyclin B

• Cyclin B is tagged for Proteosome degradation at anaphase– Tagged at N-terminus at sequence called– Destruction box– DBRP binds to Destruction box

• Guides Ubiquitin ligase to add ubiquitin molecules to Cyclin B

• Why is Cyclin B only degraded in anaphase

P

Proteinde-phosphorylase

MPF?

P

DBRP(active)

DBRP(inactive)

Ubiquitin ligase adds ubiquitin when DBRP binds to thedestruction box

Destruction box

DBRP = Destruction box recognition protein

Cyclin B

• DBRP is normally inactive and is only activated in anaphase via phosphorylation

• Possible MPF phosphorylates DBRP causing Cyclin B destruction– Binds to the destruction box– Activates ubiquitin ligase to add ubiquitin to

Cyclin B– Cyclin B then targeted to the Proteosome for

degradation

Cyclin B

• When this causes MPF inactivation – DBRP dephosphorylated by constitutive

phosphorylase• Other proteins also control MPF

– Activity doesn’t increase as Cyclin B increases

• Proteins discovered in yeast by cdc mutant complementation

Cyclin B (subunit B) Protein kinase (subunit A)

inactive

MPF}

Inactive MPF

Y15 T161

Y15 T161

Y15 T161

Y15 T161

P

P P

P

Inactive MPF

Active MPF

Wee1

CAK

cdc25

cdc2 cdc13

MPF activity

• Wee mutant small: Enters mitosis prematurely

• cdc 25 mutant long: Stays in G2 for longer

• Wee phosphorylates Y15 and inactivates MPF

• CAK (cdc2 [MPF]-activating kinase) phosphorylates T161

• cdc25 dephosphorylates Y15 and activates MPF

Cell cycle

• How is entry into S-phase controlled?

• Throughout cell cycle the protein kinase (cdc28 in sc and cdc2 in sp) binds to specific cyclins

• This changes the specificity of the protein kinase

Activity of Protein Kinase

• Cdc28-cyclins B1-4: Protein kinase activates proteins involved in early mitosis by phorphorylating them

• Cdc28-cyclins 1-3: Protein kinase activates proteins involved in initiation of DNA replication by phosphorylating them

• cdc28-cyclin 5: Phorphorylates and thus activates proteins that maintain DNA replication

How many protein kinases?

• In both yeasts only one protein kinase

• In higher eukaryotes multiple protein kinases– Active at different stages of the cell cycle

• As with yeast different cyclins