Systems Biology and Synthetic Biology.ppt

Systems Biology and Synthetic Biology

1. Biochemical Control of the
Cell Cycle
BNS230

2. Lecture programme
•Three lectures
•Aims
–Describe the cell cycle
–Discuss the importance of the cell cycle
–Discuss how the cycle is regulated

3. 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

4. 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

5. 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

6. Components of the cell cycle
•M phase
–Cell division
–Divided into six phases
•Prophase
•Prometaphase
•Metaphase
•Anaphase
•Telophase
•Cytokinesis

7. 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

8. 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

9. Components of the cell cycle
•G2-phase
–Cell checks everything is OK for cell
division
–Accumulates proteins that activate cell
division

10. 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

11. 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

12. 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

13. 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

14. 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

15. 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

16. 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

17. 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

18. 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

19. 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

20. 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

21. 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

22. 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

23. 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

24. 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

25. 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

26. 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

27. 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

28. 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

29. 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)

30. MPF
•Protein identified that causes mitosis
•Called maturation promoting factor
•MPF in all mitotic cells from yeast to
humans
•Renamed mitosis-promoting factor

31. Properties of MPF
•MPF activity changes through the cell cycle
• MPF activity appears at the G2/M interphase
• and then rapidly decrease

32. 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

33. 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)

34. 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

35. Anaphase
Telephase
Interphase
(G1-S-G2)
Prophase
Metaphase
Ubiquitin
Proteosome
Cyclin B (subunit B)
Protein kinase (subunit A)
MPF}

36. 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

37. 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

38. 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

39. 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

40. 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

41. 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

42. 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

43. 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

44. 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

45. 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