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 G 1 phase M phase G 2 phase G 0 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 minutes Plant and mammals 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°C 30°C Colonies 4 and 10 are possible cell cycle mutants. Called cell division cycle (cdc) mutants >70 cdc mutants isolated

Are the temperature sensitive mutants cdc mutants? Grow colonies at 30°C Shift temperature to 37°C Look under a microscope Colony 4: Too small; enters mitosis too early (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 and then study what the proteins do Wild type S. 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 B other 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 Protein de-phosphorylase MPF? P DBRP (active) DBRP (inactive) Ubiquitin ligase adds ubiquitin when DBRP binds to the destruction 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 P PP 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