1. BIOL 200 (Section 921) Lecture # 12; July 5, 2006 Unit 9: Cell Cycle, Cell division Readings: I. Cell cycle: ECB 2nd ed., Chapter 19, pp. 637-40. Overview of the cell cycle; Chapter 18, pp. 611-25. Good questions: 18-2, 18-3, 18-4; 18-7a, d, e; 18-10; 18-15; 18-16. ECB 1st ed., Chapter 17, pp. 547-550. [Skim pp. 551-560] Overview of the cell cycle; Chapter 18, pp. 571-581. II. DNA replication: ECB 2nd ed., Chapter 6, pp. 196-208 DNA Replication; Questions # 6-9, 6-10, 6-11 (very good). ECB 1st ed., Chapter 6, pp. 189-197 DNA Replication; Questions # 6-14, 6- 15, 6-16 (very good) III. Chromosomes and Mitosis: ECB 2nd ed., Chapter 19 pp 639-55 for Mitosis. Good questions: 19-3, 19-4; 19-5; 19-8; 19-10; 19-12; 19-14. ECB 1st ed., Chapter 8, p 249-50 Telomeres: Specialized structures ensure that chromosomes replicate efficiently; Chapter 17 pp 551-562 for Mitosis

2. Learning objectives I. Introduction and Overview of Cell Cycle; Regulation of cell cycle by CDK-cyclin Understand overall organization of the cell cycle and its relation to cell division (mitosis and cytokinesis): Be able to list cell cycle stages in order and indicate the location of the two major cell cycle control points. Give examples of discrete and continuous cell cycle processes. Understand the checkpoint concept and how this allows Integration of continuous and discrete cell cycle processes Understand the role of CDK-cyclin in regulation of cell cycle processes. Be able to explain how the CDK complex is regulated by protein phosphorylation, and by proteolysis. II. DNA Replication Explain what is meant by semi-conservative replication of DNA. Explain how DNA replication occurs in eukaryotes through the independent operation of many replicons. Describe the roles of the following in DNA replication: Helicase, DNA polymerase, primer, Okazaki fragments, ligase. Explain the terms lagging and leading strand and the significance of these terms for DNA replication. Understand the relation between chromatids and chromosomes in both pre- and post-replication stages Explain and understand replicon and replication fork. III. Chromosomes and Mitosis Understand why telomeres are important and how telomere extension occurs List the stages of mitosis in order and explain what is happening to the cytoskeleton, the mitotic spindle, the nuclear envelope and the chromosomes during this process. Explain how chromosomes are moved to the poles at mitosis. What are kinetochores and what are they good for.

3. Cell cycle Cells arise from pre-existing cells The reproduction of cells takes place in an orderly fashion and is genetically regulated Cells reproduce by a process called the cell cycle which show (i) cell growth and chromosome replication, (ii) chromosome segregation and (iii) cell division Each cell type has its own timing and built-in memory For example, cell cycle time varies from about 0.5 hours in early frog embryo cells to about 1 year in human liver cells

4. Four phases of the cell cycle [Fig. 18-2] M phase includes nuclear division and cytokinesis G1 – the interval between M and S phases S phase – replication G2 – the interval between S phase and mitosis

5. A central control system (like a washing machine) triggers the major processes of the cell cycle [Fig. 18-3] Like a clothes- washing machine, there is a feedback at each stage The next stage can not begin until the previous one has ended This is a system of checkpoints

6. Two major checkpoints in the cell cycle There is a checkpoint in G1 for cell size. If the cell has not grown sufficiently, it will be held in G1 until that happens Similarly, there is a checkpoint in G2 that ensures replication is complete before mitosis begins Checkpoints also take into account signals from other cells and environment. ‘START’ ‘COMMITMENT TO DIVN.’

7. Xenopus egg, does not divide unless activated [Fig. 18-8]

8. microinjection of cytoplasm extract into egg activates mitosis [Fig. 18-9] CONCLUSION: Some kind of cytoplasmic factor promotes cell division – called m phase promoting factor (MPF) Inject cytoplasm from m phase cell Promotes mitosis Inject cytoplasm From interphase cell No mitosis, stays in interphase

9. Cyclin-CDK complex [Fig. 18-5] Cyclin- amount varies during cell cycle Cyclin-dependent kinase (Cdk)

10. The major Cyclins and Cdks [Table 18-2] -------------------------------------------------------- Cyclin-CdkCyclinCdk partner Complex ----------------------------------------- G1-Cdkcyclin DCdk4, Cdk6 G1/S-Cdkcyclin ECdk2 S-Cdkcyclin ACdk2 M-Cdkcyclin BCdk1 --------------------------------------------------------

11. Changes in [M-cyclin] and M-Cdk activity during the cell cycle [Fig. 18-6]

12. Factors affecting the activity of Cdks 1.Cyclin degradation by ubiquitination 2.Phosphorylation and dephosphorylation 3.Positive feedback 4.Cdk inhibitor proteins

13. 18_07_cyclin_degradat.jpg Cyclin degradation by ubiquitination inhibits the activity of Cdks [Fig. 18-7]

14. Phosphorylation and Dephosphorylation Cell cycle control is regulated by phosphorylation and dephosphorylation Protein kinases and phosphatases form a switch Protein kinases that control cell cycle are present throughout the cell cycle. Their activity rises and falls. A second set of proteins CYCLINS have no enzyme activity themselves. They bind to kinases and activate them. The kinases of the cell cycle control system are known as cyclin-depedent protein kinases or Cdks.

15. Protein phosphorylation [Fig. 4-41] kinase adds Phosphatase removes phosphate group

16. Protein phosphorylation acts as a switch to modify protein activity [Fig. 4-41]

17. 18_11_M_Cdk_active.jpg Selective phosphorylation and dephosphorylation activate M-Cdk [Fig. 18-11] Thr 14, Tyr 15 Thr 161

18. Fig. 18-12: CDK phosphorylates activating phosphatase: further activation of CDK via a positive feedback loop.

19. 18_13_Cdks_cyclins.jpg Different cyclin-Cdk complexes trigger different events in cell cycle [Fig. 18-13]

20. M-Cdk effects 1.M-Cdk contains a single protein kinase, which triggers mitosis 2.It phosphorylates MAPs and causes cytoskeleton to reorganize – forming the spindle 3.It posphorylates the nuclear lamina and causes the nuclear envelope to break down. 4.It phosphorylates non-histone proteins and causes chromosome condensation

21. 18_15_cell_cycle_G1.jpg DNA damage arrests the Cell cycle in G1 [Fig. 18-15]

22. 18_17_arrest_checkpt.jpg The cell-cycle control system can arrest the cycle at various checkpoints [Fig. 18-17] Cell below critical size Cell below critical size

23. DNA replication Learning objectives Explain what is meant by semi-conservative replication of DNA. Explain how DNA replication occurs in eukaryotes through the independent operation of many replicons. Describe the roles of the following in DNA replication: Helicase, DNA polymerase, primer, Okazaki fragments, ligase. Explain the terms lagging and leading strand and the significance of these terms for DNA replication. Understand the relation between chromatids and chromosomes in both pre- and post-replication stages Explain and understand replicon and replication fork

24. Semiconservative replication of DNA: Each daughter strand has one DNA template from parental strand [Fig. 6-3] “S” PHASE

25. 06_05_replic.origin.jpg Replication initiator proteins open up a DNA double helix at its replication origin [Fig. 6-5]

26. 06_09_Replic.forks.jpg Replication fork moves away in both directions

27. 06_10_5prime_3prime.jpg DNA synthesis in the 5’ →3” direction

28. 06_11_oppositepolarity.jpg The two newly synthesized DNA strands have opposite polarity

29. 06_12_asymmetrical.jpg DNA replication forks are asymmetrical [Fig. 6-12]

30. 06_14_polymerase2.jpg DNA POLYMERASE CATALYZE BOTH DNA REPLICATION AND EDITING (PROOFREADING) [Fig. 6-14]

31. 06_16_lagging strand.jpg On the lagging strand, DNA is synthesized in fragments [Fig. 6-16]

32. 06_17_group proteins.jpg A group of proteins act together at a replication fork

33. A protein machine in DNA replication Key Players Leading strand DNA Lagging strand DNA DNA Polymerase III Helicase RNA Primase and RNA Primers Okazaki Fragments Ligase Single-stranded DNA binding proteins

34. Becker et al. The World of the Cell]

35. Chromosomes and Mitosis: Learning objectives Understand why telomeres are important and how telomere extension occurs List the stages of mitosis in order and explain what is happening to the cytoskeleton, the mitotic spindle, the nuclear envelope and the chromosomes during this process. Explain how chromosomes are moved to the poles at mitosis. What are kinetochores and what are they good for.

36. 3 DNA sequences needed to produce a eukaryotic chromosome [Fig. 5-18] 1. telomere 2. origin of replication 3. centromere

37. 06_18_telomeres.jpg Telomeres allow the completion of DNA synthesis at the ends of Chromosomes [Fig. 6-18] Telomerase contains a Short piece of RNA which acts as a primer for DNA synthesis

38. 19_04_mediate_M.jpg The cytoskeleton is involved in both mitosis and cytokinesis

39. Mitosis separates sister chromatids into daughter chromosomes [Fig. 19-6]

40. Panel 19-1: prophase

41. Mitotic chromosome [Fig. 19-3] 2 chromatids centromere What keeps the 2 chromatids together?

42. Cohesins hold sister chromatids together. Condensins help pack chromatin into chromosomes [Fig. 19-3] Where would you expect to find cohesins in this picture?

43. Fig. 19-5: centrosomes duplicate to form mitotic spindle poles. centrosome duplication Mitotic spindle poles

44. Fig. 19-7: Mitotic spindle Selective stabilization of interpolar MT prophase

45. Panel 19-1: prometaphase Dynamic instability- MT’s shoot out of spindle poles.

46. Fig. 17-9: Kinetochore is protein complex at centromere region of chromosome MT bind here

47. Panel 19-1: Metaphase

48. Fig. 19-13: Mitotic spindle at metaphase Kinetochore microtubules Interpolar microtubules

49. Panel 19-1: Anaphase

50. Fig. 19-17: Anaphase A-sister chromosomes separate by kinetochore MT shortening

51. Fig. 19-17: Anaphase B-poles move apart by polar MTs sliding past each other motors slide polar MTs

52. 19_16_APC_triggers.jpg The anaphase promoting factor (APC) triggers the separation of sister chromatids by promoting the destruction of cohesins [Fig. 19-16]

53. Fig. 19-19: cleavage furrow made by actin- myosin ring pulling PM in to divide daughter cells

54. 19_20_contractile_ring.jpg Cytokinesis-division of cytoplasm due to actin-myosin contractile ring

55. Fig. 18-38: Golgi derived vesicles make a new cell wall in plants mitotic spindle Golgi Cell plate Decondensing chromosomes of daughter nuclei

56. Fig. 19-22: cell plate forms during cytokinesis in plants