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M267 Lecture 4, Cell Cycle February 27, 2006

M267 Lecture 4, Cell Cycle February 27, 2006. Cohesins. cyclin deg. cdc2/28. G 2. Prophase. Metaphase. Anaphase. Telophase. G 1. - p34 cdc2 cyB. +p34 cdc2 cyB. 0 15 30 50 0 15 30 50. min. min. interphase. mitotic. M extract. I extract. buffer.

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M267 Lecture 4, Cell Cycle February 27, 2006

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  1. M267 Lecture 4, Cell Cycle February 27, 2006

  2. Cohesins cyclin deg. cdc2/28 G2 Prophase Metaphase Anaphase Telophase G1

  3. -p34cdc2cyB +p34cdc2cyB 0 15 30 50 0 15 30 50 min min interphase mitotic

  4. M extract I extract buffer Time 0 6 12 20 25 30 35 40 60

  5. cyclin B cyclin B cdc2 p cdc2 p cdc25 p p p p cyclin B cyclin B + cdc2 p cdc2 cyclin B sythesis p p

  6. cyclin B protein X cyclin B cdc2 p cdc2 p cdc25 p p p p protein X p p cyclin B cyclin B protease + cdc2 p cdc2 cyclin B sythesis p p

  7. Ub Ub Ub Ub p p APC proteasome Cyclin B Cyclin B Cyclin B Ub Ub Ub Ub

  8. Cln1- Cln2- Cln3- G1 M G2 S

  9. Presence of methionine Cln1- Cln2- Cln3- G1 M G2 S Met-cln2 (met repressible) Gal-clb2 (gal inducible)

  10. Cyclin B RNA Cyclin B protein H1 kinase activitry These cells do not express any G1 cyclins (clns). Consequently, they arrest in G1. They can accumulate cyclin B mRNA in response to galactose. Although cyclin B mRNA can be made, cyclin B protein cannot be made – cyclin B protein continues to be degraded. Since no cyclin is available to activate cdc28, no CDK activity (e.g. histone phosphorylation activity) is present. + methionine, + galactose

  11. Cyclin B RNA Cyclin B protein H1 kinase activitry If one removes methionine, Cln2 (the G1 cyclin) is made. Now cyclin B protein can be made. Now that cyclins are made, the CDK catalytic subunit can be activated, and CDK activity (histone phosphorylation) can occur

  12. APC mediated destruction machinery for cyclin B Cyclin Oscillations in yeast CLN1, CLN2 and CLN3 associated kinases are absent in early G1, reach peak levels at the G1-S transition, and decline as cells enter G2. These kinases not only promote budding and DNA synthesis, but also inactivate CLB cyclin proteolysis. Activation of the mitotic kinase during G2 causes repression of CLN1, CLN2 and CLN3 synthesis and onset of M phase and ultimately leads to the reactivation of CLB proteolysis. Cyclin B proteolysis then persists during G1 until the reactivation of CLN cyclins

  13. Cyclin B RNA Cyclin B protein H1 kinase activitry What would happen to CLB2 synthesis if the cyclosome was “broken”? Gal promoter CLB2 protein b-gal protein

  14. pGAL-CLB2 -GAL +GAL WT Cdc16-123 Cdc23-304 Cse1-22

  15. Cse1 cdc16 cdc27 cdc23 The cyclosome, the “machine” that ubiquitinates cyclin B, and leads to cyclin B desruction

  16. Express a non-degradable Cyclin B Cells arrest here Cells do not arrest at anaphase, even though cyclin B is present.

  17. Inhibit the activity of the cyclosome/APC Cells arrest at anaphase

  18. Inhibit the activity of the cyclosome/APC APC directed degradation of some other protein – before cyclin is degraded – must be necessary for sister chromatid separation; i.e., for the metaphase to anaphase transition to occur. Degradation of cyclin B must be necessary for a subsequent step; for the transition from telophase to G1. ? Cells arrest at anaphase

  19. Cut 2 accumulates in anaphase in APC mutants Expression of a non-degradable form of Cut 2 causes arrest in anaphase.

  20. Cohesin, holds sister chromatids together Separase, a protease that cleaves cohesins, permitting chromosome separation (i.e., anaphase) to occur When securins are destroyed, separase is activated, the cohesins are cleaved, and the sister chromatids can separate in anaphase. Securins; inhibitors of the separase enzyme.

  21. securin Cohesins, separase and securins

  22. Pds1 (securin) Clb2

  23. Hct1 Cdc20 securin Cohesins, separase and securins

  24. Pds1/Cut2/securin

  25. 1 2 3 4 5 6 Time (min) 0 15 30 0 15 30 60 1, 3, 5: w.t. cdc20. 2, 4, 6: mutant cdc20.

  26. APC APC cdc20 cdh1 proteasome proteasome Substrate 1 Substrate 2 AT M/G1 AT ANAPHASE D-BOX (CUT2) Target RXXLXXXXN Target KENXXXN Target

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