Eukaryotic cell cycle

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

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