Eukaryotic cell cycle
- S: Synthesis
- Replication
- M Mitosis + Cytokinesis
- Nuclear Division -> Mitosis
- Cytoplasmic Division -> Cytokinesis
- G Gap phases
- Regulatory and Delay steps
- in human embryonic cells
- 24 hr cell cycle
- 23 hours Interphase
- 1 hr mitosis
- 24 hr cell cycle
- Stages of the Cell cycle
- G1
- before replication and division
- monitors environment of the cell -> ensures factors are appropriate
- chromosomes are each a single chromatid
- S
- replication of chromosomes
- G2
- After replication and before M phase
- checks the correct duplication of the chromosomes
- chromosomes consists of a pair of sister chromatids
- M
- Nuclear and cytoplasmic division
- G1
- Phases of Mitosis
- Prophase
- condensation of chromosomes
- Metaphase
- nuclear envelope breakdown, assembly of spindle, attachment of chromatids on spindle, alignment of chromatids
- Anaphase
- separation of sister chromatids
- push and pull of microtubules -> migration into opposite poles
- Telophase
- decondensation of the chromosomes
- clustering of the poles
- Prophase
- cdc mutants
- cell division cycle mutants
- mutants, that are halted within specific points of the cell cycle
- each mutant defines a gene, whose function is required once per cell cycle
- conditional, but only active at a non elevated temperature
- examples
- wee mutants
- cell division occur at a reduced size than normal
- cell cycle progressed prematurely; LINK
- deficit of cdc25 or excess wee1
- elongated
- fail to divide but continue to grow
- deficit of wee1 or excess cdc25
- fail to divide but continue to grow
- wee mutants
- cycline dependent kinases
- interact with cyclins
- need to be phosphorylated at specific residues for activation or inhibition (partial)
- requires cyclin binding for initial partial activation
- in humans -> several
- in yeast -> one
- Cycline D – CDK4 / 6 – G1
- Cycline E / E – CDK2 – G1/S
- Cycline A /B – CDK1 S / G2
- Methods to identify interacting Proteins
- high copy suppression
- Mutation of both interacting partners lead to wild-type phenotype LINK
- synthetic lethality
- two hybrid system
- transformation of yeast strain with both prey and bait plasmids
- high copy suppression
- Cdk regulation
- partial, initial activator -> Cyclin (e.g cdk1 – cyclin B)
- shifts the T loop / rearrangement of 3 structure of the interaction site
- CAK – CDK activating kinase
- phosphorylates the Y15 residue in the interacting region of the kinase
- wee1 – inhibitory phosphorylating kinase
- phosphorylates at the T161 site
- cdc25 – activating phosphorylating protein
- removes the inhibitory phosphorylation (made by wee1)
- CKI – CDK inhibitor
- partial, initial activator -> Cyclin (e.g cdk1 – cyclin B)
- classes of cdk inhibtors – > CKI
- p16^ink4
- cip/kip
- classes of ubiquitin ligase
- Hect E3
- Ring E3 with one subunit
- mdm2
- Ring E3 with multiple subunits
- apc, spf
- the proteosome of the ubquitin proteosome system
- made up of three subunits
- 19s (2X)
- 20s (1X)
- the lid
- 19s
- ubiquitin binding proteins
- de-ubiquitylation enzymes
- AAA ATpase ring
- 19s
- made up of three subunits
- mitotic entry requires cdk-cyclin B
- mitotic exit requires degradation of cyclin B
- experimental proof
- prepare a xenopus extract; cell cycle halted in either anaphase or interphase
- prepare radio labeled cyclin B
- add to extract
- separate on gel based on cell cycle stage and time SDS Page and Autoradiography
- example
- anaphase; time – 0 min
- cyclin B band visible
- anaphase time – 30 min
- cyclin B band invisible
- interphase time – 0min
- cyclin B band visible
- interphase time – 30min
- cyclin B band visible
- anaphase; time – 0 min
- OR radio labeled cyclin B / ubiquitin
- Analysis of the metaphase-anaphase transition
- cyclin B degradation essential
- RNAse treated extract + cyclin B (wild type)
- tubulin unseen after 80 min; daughter cells visible
- RNAse treated extract – nondegradable (mutant) cyclin B
- tubulin still visible after 80 min; DNA still condensed
- RNAse treated extract + cyclin B (wild type)
- APC
- Anaphase promoting complex
- DB – Destruction peptide box – recognition peptide sequence -> competitve inhibitor of APC
- Untreated extract + DB cyclin B
- the lower the amount of DB, the further the separation of the mitotic spindle
- Anaphase promoting complex
- cyclin B degradation essential
- Cohesin
- mediates sister chromatid separation