Overview of Cell Cycle

1. Overview of Cell Cycle • Interphase: G1, S, and G2 phase • (The end of cell division to The start of next cell division) • Mitosis: M phase • (The period of cell divided into two daughter cells)

2. Synthesis at S Phase • S phase: synthetic period • (DNA is specifically & largely replicated) • (Genetic information is duplicated) • Interphase: • (Continuous increase of RNA & protein) 2n DNA to 4n DNA

3. Commitment to Cell Division • G1 phase: START • (Restriction/ Commitment point beyond S phase) • (Ensure the ability of cell to replicate chromosome) • G2 phase: • (Restriction/ Commitment point beyond Mitosis phase) • (Ensure the ability of cell to divide) • G1>G2

4. Orderly Progress of Cell Cycle • Make a decision • G1 restriction point: • (Nutrient supply is enough?) • (Cell mass is sufficient to support division?) • S phase: • (Replication apparatus is ready to synthesis DNA? ) • (The newly synthesized DNA is complete?) • M phase: • (The newly synthesized DNA is complete?) • (Duplicated chromosome is properly segregated?) Checkpoint: A control loop that makes the initiation of one event in the cell cycle dependent on the successful completion of earlier event.

5. Checkpoints at Cell Cycle: • S phase: • Loss of the integrity of the DNA • (DNA damage & break) • Mitosis phase: • Incompletion of chromosome segregation • (Unattached kinetochore)

6. Controlled Events of Cell Cycle • Regulatory & Cyclical Events Control Transitions between phases: • The newly synthesized of proteins/ the degradation of existed proteins • Activation/ inactivation of molecules • G1 phase: • Phosphorylation of RB • S phase: • S phase activator/ protein kinase • M phase: • M phase protein kinase

7. Identify S phase Inducer • Cell Fusion Experiment: • Characterizes the activators of each phases • Mix the cells in the presence of chemical of viral agents • The hybrid cell contains two nuclei in a common cytoplasm (heterokaryon)

8. Identify M phase Inducer (MPF) • Xenopus laevis oocytes exp: • Arrested oocytes(G2 phase) • → Ovulation • → Arrested egg (M phase) • The cytoplasm of arrested egg can cause the arrested oocytes to enter meiosis • Maturation promoting factor (MPF) • (Disaggregation of nuclear envelope) • (Condensation of chromosome) • (Spindle formation) • (Phosphorylation a variety of proteins → kinase)

9. MPF is the Cdk/Cyclin • MPF: • Cdc2: a catalytic subunit of serine/theronine kinase activity • Cyclin: a regulatory subunit promotes Cdc2 phosphorylated appropriate substrates • Cyclin activates Cdc2 by a binding-induced conformational change • Two Forms of MPF: • Cdc2-cyclin A • Cdc2-cyclin B • Regulation of MPF: • Cdc2 constantly expressed/ Cyclin is degraded • Cdc2 is modified by kinase (+p) • Cyclin is regulated by proteolysis • Cdc2 is modified by phosphatase (-p)

10. Yeast Is a Model System for Analyzing Cell Cycle S. pombelengthens and then divides with a septum, while S. cerevisiaebuds during a cycle in which G2 is absent and M occupies the greatest part.

11. Identify Cell Cycle Mutants in S. pombe

12. Cdc Genes in the Cell Cycle of S. pombe

13. Different Forms of Cdc2-Cyclin in the Stages of Cell Cycle • M phase: • Cdc2/Cdc13 (Try-15 –p; Thr-161 +p) • At late mitosis: Cdc13 is degraded • START: • Cdc2/Cdc13 (Try-15 + p; Thr-161 +p) • S to M phase: • Cdc2/Cdc13 is activated (Try-15 – p; Thr-161 +p) by dephosphorylation

14. Identify Cell Cycle Mutants in S. cerevisiae Haploid yeast cells of either a or a mating type may reproduce by a mitotic cycle. Cells of opposite type may mate to form an a/a diploid. The diploid may sporulate to generate haploid spores of both a and a types. Cdc28 is the Cdc2 of S. cerevisiae

15. Cell Cycle of S. cerevisiae • Chromosome cycle: • Chromosome duplication • G1-S phase • Cdc8 mutant • Centrosome cycle: • Spindle pole body segregation • Organize microtubules to allow chromosome segregation • Cdc31 mutant • Cytoplasmic phase: • Nuclear migration into the bud • Cdc24 mutant

16. Cell Cycle Control in S. cerevisiae

17. The Cdc25 Phosphatase & Wee1 Kinase Control Cdc2 Activity • Cdc25: • Phosphatase • Remove inhibitory phosphate from Tyr-15 of Cdc2 • Wee1: • Kinase • Phosphorylate Tyr-15 of Cdc25 S. pombe

18. Cdc18 Controls S phase Rum1 Inactivates MPF S. cerevisiae

19. DNA Damage Triggers a Checkpoint Prevents a defect in genetic information Blocks the cell cycle progression Damaged DNA is repaired DNA damage → ATMkinase → phosphorylates Chk2 → phosphorylates/ inactivate Cdc25

20. DNA Damage Triggers the G2 Checkpoint DNA damage → ATMkinase → phosphorylates Chk2 → phosphorylates/ inactivate Cdc25

21. Checkpoints function at each stage of the cell cycle in S. cerevisiae DNA damage → Sensor → Transducer → Effector

22. Animal Cell Cycle Is Controlled by Many Cdk-Cyclin Complexes. • Many dimeric kinase complex (Cdk-cyclin) function in animal cells • Cdk2 & Cdk4: • Form a complex with G1 cyclins

23. Control of Mitosis in Animal Cells Requires Phosphorylation and Dephosphorylation of MPF • Cdc2: • Thr-161 is phosphorylated by Cdc2-activating kinase (CAK) = Wee1 • Cdc25: • Phosphatase • Remove inhibitory phosphate from Tyr-15 of Cdc2

24. Several Cdk-Cyclin Complexes Are Active at G1 and S Phases • Cdk2, Cdk4, Cdk5: • Thr-161 is phosphorylated by Cdc2-activating kinase (CAK) = Wee1 • D cyclins: • Active at G0/G1 phase • Controlled by synthesis/ degradation • E cyclins: • Active at G1/S phase • Controlled by synthesis/ degradation • A cyclins: • Active at S phase and G2/M phases (only) • Controlled by synthesis/ degradation

25. Which Protein Is Activated by Cdk-Cyclin Complex? • RB: • Is a target of cdk-G1 cyclin (D) complexes • Is a tumor suppressor • RB-E2F → transcription inhibited • RB-p E2F → transcription activated • Cdk-cyclin complex phosphorylates RB

26. Cdk Inhibitors Represent Alternative Ways to Control Cell Cycle • Cdk-cyclin inhibitors: • CKI • CKI has its specificity to different cdk-cyclin • Binds to cdk-cyclin • Interferes with the phosphorylation of RB

27. Target of CKI to Protein Degradation Controls G1/S Progression • Sic1: • A CKI in S. cerevisiae • Binds to CDC28-CLB in G1 phase • Sic1 is degraded when cells enter S phase • Sic1 is degraded by proteolysis • SCF (Cdc53-Skp1-Cdc4) E3 ligase confers the proteolysis of Sic1 • p27, p21, and cyclinE are also substrates of SCF E3 ligase

28. Protein Degradation Also Regulates G2/M Progression • Cyclin A: • Is degraded in metaphase • Cyclin B: • Is degraded in anaphase • Pds1p: • Is degraded in anaphase • Ensures sister chromatids to seperate

29. Anaphase Promoting Complex/ Cyclosome (APC/C)Regulates Proteolysis at G2/M Phase • E3 ubiquitin ligase: • Activated by phosphorylation • Degrade: • B-type cyclin, Pds1p, and the molecules unnecessary for mitosis • Nucleotide synthesis enzyme • DNA replication complex • subunit

30. The Proteolytic Activity of APC/C Ensure the Proper Segregation of Sister Chromatids • Pds1p/Securin: • Is degraded by APC/C • Esp1/Separin : • Is activated by degradation of securin

31. The APC/C Is Maintained at Inactive State until All Kinetochores Are Attached • Mad proteins: • Inhibit the activity of APC/C by binding to CDC20 • Bub proteins : • Inhibit the activity of APC/C by binding to CDH1

32. Exit from Mitosis Is Controlled by Cdc14 • Cdc14: • A phosphatase dephosphorylates Cdh1 → activate APC/C • A phosphatase dephosphorylates Sic1 → inactivate mitotic cyclins • In nucleolus at interphase

33. Cell Structure Is Reorganized at Mitosis

34. Spindle Assembly & Microtubule Nucleation

35. Overview of Cell Cycle • Protein synthesis/ degradation • Transcription/ ubiquitin E3 ligase • Activate/ Inactivate • Post-translational modifications • Coordinated events through cell cycle • Conserved machinery during evolution