2. Gihan E-H Gawish, MSc, PhD Ass. Professor Molecular Genetics and Clinical Biochemistry Molecular Genetics and Clinical BiochemistryKSU 7 th WEEK Cell Cycle

3. THE CELL CYCLE  Cell Cycle  Introduction to cell cycle  CYCLINS and cdk’s  CELL CYCLE CHECKPOINTS  Mechanisms of Carcinogenesis  TUMOR SUPPRESSOR GENES  ONCOGENES Genetic and molecular studies in diverse biological systems have resulted in identification and characterization of the cell cycle machinery

4. Mitotic spindle DNA replication Chiasmata Dynamic instabilty Cdc mutantsCell-cycle control Maturation-promoting factor Regulation of Cdc2 Cyclin characterization Checkpoint control p53 The mitotic checkpoint The APC and proteolysis SCF and F-box proteins The restriction point Yeast centromeres Cell-cycle conservation Replication origins Retinoblastoma/E2F Body-plan regulation A new class of cyclins CDK inhibitors Sister-chromatid cohesion

5. Living cells go through a series of stages known as the cell cycle. The cells grow, copy their chromosomes, and then divide to form new cells. G1 phase. The cell grows. S phase. The cell makes copies of its chromosomes. Each chromosome now consists of two sister chromatids. G2 phase. The cell checks the duplicated chromosomes and gets ready to divide. M phase. The cell separates the copied chromosomes to form two full sets (mitosis) and the cell divides into two new cells (cytokinesis). Living cells go through a series of stages known as the cell cycle. The cells grow, copy their chromosomes, and then divide to form new cells. G1 phase. The cell grows. S phase. The cell makes copies of its chromosomes. Each chromosome now consists of two sister chromatids. G2 phase. The cell checks the duplicated chromosomes and gets ready to divide. M phase. The cell separates the copied chromosomes to form two full sets (mitosis) and the cell divides into two new cells (cytokinesis).

6. Mitosis is used to produce daughter cells that are genetically identical to the parent cells. The cell copies - or 'replicates' - its chromosomes, and then splits the copied chromosomes equally to make sure that each daughter cell has a full set Meiosis is used to make special cells - sperm cells and egg cells - that have half the normal number of chromosomes. It reduces the number from 23 pairs of chromosomes to 23 single chromosomes. The cell copies its chromosomes, but then separates the 23 pairs to ensure that each daughter cell has only one copy of each chromosome. A second division that divides each daughter cell again to produce four daughter cells. Animation Animation Meioses Animation Animation Meioses Animation Animation Mitosis Animation Animation Mitosis

7. A diagram of a cell ready for mitosis. The copied chromosomes consist of two chromatids joined at the centromere Mitosis is a way of making more cells that are genetically the same as the parent cell. It plays an important part in the development of embryos, and it is important for the growth and development of our bodies as well. Mitosis produces new cells, and replaces cells that are old, lost or damaged. In mitosis a cell divides to form two identical daughter cells. It is important that the daughter cells have a copy of every chromosome, so the process involves copying the chromosomes first and then carefully separating the copies to give each new cell a full set. Before mitosis, the chromosomes are copied. They then coil up, and each chromosome looks like a letter X in the nucleus of the cell. The chromosomes now consist of two sister chromatids. Mitosis separates these chromatids, so that each new cell has a copy of every chromosome Mitosis is a way of making more cells that are genetically the same as the parent cell. It plays an important part in the development of embryos, and it is important for the growth and development of our bodies as well. Mitosis produces new cells, and replaces cells that are old, lost or damaged. In mitosis a cell divides to form two identical daughter cells. It is important that the daughter cells have a copy of every chromosome, so the process involves copying the chromosomes first and then carefully separating the copies to give each new cell a full set. Before mitosis, the chromosomes are copied. They then coil up, and each chromosome looks like a letter X in the nucleus of the cell. The chromosomes now consist of two sister chromatids. Mitosis separates these chromatids, so that each new cell has a copy of every chromosome

8. The process of mitosis involves a number of different stages

9. Mitosis Prophase: The replicated chromosomes condense and the mitotic spindle begins to assemble outside the nucleus. Prometaphase: The membrane surrounding the nucleus (nuclear envelope) breaks down and allows the mitotic spindle to contact the chromosomes. Metaphase: All the chromosomes are gathered at the center of the mitotic spindle Anaphase: The chromosomes are split apart and pulled to opposite sides of the cell Telophase: The nuclear envelope reassembles around the two new sets of separated chromosomes to form two nuclei.

10. Cytokinesis  The second feature of M phase: It is the time in which the other components of the cell— membranes, cytoskeleton, organelles, and soluble proteins—are distributed to the two daughter cells. This is the final task that a cell must complete to finish its reproduction

11. Meiosis  The cells in our bodies contain 23 pairs of chromosomes - giving us 46 chromosomes in total.  Sperm cells and egg cells contain 23 single chromosomes, half the normal number, and are made by a special form of cell division called meiosis.  Meiosis separates the pairs of matching (or 'homologous') chromosomes, so that sperm cells and egg cells have only one copy of each.  That way, when an egg cell fuses with a sperm cell, the fertilised egg has a full set: that is, two copies of every chromosome.  Meiosis involves two cell divisions: Meiosis I and Meiosis II.

12. Meiosis I separates the matching - or 'homologous' - pairs of chromosomes. Each daughter cell receives a mix of chromosomes from the two sets in the parent cell. The chromosomes in each matching pair swap some genetic material before they are parted in a process called crossing over. These processes produce new combinations of genes in the sperm cells and egg cells. Meiosis I separates the matching - or 'homologous' - pairs of chromosomes. Each daughter cell receives a mix of chromosomes from the two sets in the parent cell. The chromosomes in each matching pair swap some genetic material before they are parted in a process called crossing over. These processes produce new combinations of genes in the sperm cells and egg cells.

14.  CYCLIN DEPENDENT KINASES (cdk)  CYCLINS  Regulators of CYCLIN/cdk  Activating Phosphatases  Inhibitory Kinases  Non-kinase inhibitors

15. tyr15-P P-thr161 thr14-P e.g. cdk1 (= cdc2) - protein kinase - binds to cyclin - kinase domain - regulatory domain - present throughout cell cycle

16. - no intrinsic enzymatic activity - binds cdk - synthesized and degraded each cycle - essential component for cdk activity e.g. Cyclin B

17. tyr15-P P-thr161 thr14-P cdk1 (cdc2) cyclin B Regulated by: -tyr15 phosphorylation inhibitory kinases activating phosphatases -direct interaction inhibitory proteins e.g. p21, p27, p57 p16, p15, p18,p19

18.  budding yeast (S cerevisiae) = cdc28  fission yeast (S pombe) = cdc2  man = cdc2 homolog; p34 cdk1 = G2/M transition cdk2 = G1/S transition u budding yeast (S cerevisiae) = cdc28 u fission yeast (S pombe) = cdc2 (S form) u man = cdk2; p33 - PSALRE motif

19.  CYCLIN B = G2/M with cdc2; =cdc13 ; =CLB1-4  CYCLIN A = S + G2 with cdk2; cig2 ; =CLB5,6  CYCLIN E = G1/S with cdk2; cig1 ?puc1; =CLN1,2,3  CYCLIN D = G1 with cdk4,5,6; in yeast (early vs late G1)  CYCLIN C = present through cycle: CTD of RNA pol II  CYCLIN F = peaks like A; cyclin stability and proteolysis  CYCLIN G = induced 3 hr growth stim and by p53: function  CYCLIN H = part of CAK (with cdk7)  CYCLIN J = sorry, it exists

20. G1 S G2 M CYCLIN B/ cdc2 CYCLIN A/ cdk2 CYCLIN E / cdk2 CYCLIN D / cdk4,5,6

21. Cell Cycle Regulators and Cancer

22. G 1 cyclin-CDK complexes become active to prepare the cell for S phase promoting the expression of transcription factors that in turn promote the expression of S cyclins and of enzymes required for DNA replication. also promote the degradation of molecules that function as S phase inhibitors by targeting them for ubiquitination. G 1 cyclin-CDK complexes become active to prepare the cell for S phase promoting the expression of transcription factors that in turn promote the expression of S cyclins and of enzymes required for DNA replication. also promote the degradation of molecules that function as S phase inhibitors by targeting them for ubiquitination.

23. Active S cyclin-CDK complexes phosphorylate proteins that make up the pre-replication complexes assembled during G 1 phase on DNA replication origins. The phosphorylation serves two purposes: to activate each already-assembled pre-replication complex to prevent new complexes from forming. This ensures that every portion of the cell's genome will be replicated once and only once. Active S cyclin-CDK complexes phosphorylate proteins that make up the pre-replication complexes assembled during G 1 phase on DNA replication origins. The phosphorylation serves two purposes: to activate each already-assembled pre-replication complex to prevent new complexes from forming. This ensures that every portion of the cell's genome will be replicated once and only once.

24. Mitotic cyclin-CDK complexes synthesized but inactivated during S and G 2 phases promote the initiation of mitosis by stimulating downstream proteins involved in chromosome condensation and mitotic spindle assembly. A critical complex activated during this process is anaphase- promoting complex (APC): promotes degradation of structural proteins associated with the chromosomal kinetochore APC also targets the mitotic cyclins for degradation, ensuring that telophase and cytokinesis can proceed. Mitotic cyclin-CDK complexes synthesized but inactivated during S and G 2 phases promote the initiation of mitosis by stimulating downstream proteins involved in chromosome condensation and mitotic spindle assembly. A critical complex activated during this process is anaphase- promoting complex (APC): promotes degradation of structural proteins associated with the chromosomal kinetochore APC also targets the mitotic cyclins for degradation, ensuring that telophase and cytokinesis can proceed.

25. Two families of genes: prevent the progression of the cell cycle.  the cip/kip family  INK4a/ARF (Inhibitor of Kinase 4/Alternative Reading Frame) Because these genes are instrumental in prevention of tumor formation, they are known as tumor suppressors. Two families of genes: prevent the progression of the cell cycle.  the cip/kip family  INK4a/ARF (Inhibitor of Kinase 4/Alternative Reading Frame) Because these genes are instrumental in prevention of tumor formation, they are known as tumor suppressors.

26.  includes the genes p21, p27 and p57.  They halt cell cycle in G 1 phase, by binding to, and inactivating, cyclin-CDK complexes.  p21 is activated by p53  p27 is activated by Transforming Growth Factor β (TGF β), a growth inhibitor.TGF β  includes the genes p21, p27 and p57.  They halt cell cycle in G 1 phase, by binding to, and inactivating, cyclin-CDK complexes.  p21 is activated by p53  p27 is activated by Transforming Growth Factor β (TGF β), a growth inhibitor.TGF β

27. 1.p16INK4a, which binds to CDK4 and arrests the cell cycle in G 1 phase 2.p14arf which prevents p53 degradation. And the amount of chromosomes are able to double at the same rate as in S phase. 1.p16INK4a, which binds to CDK4 and arrests the cell cycle in G 1 phase 2.p14arf which prevents p53 degradation. And the amount of chromosomes are able to double at the same rate as in S phase.

28. Three main checkpoints in the cell cycle 3. 1.Is cell the correct size? Is DNA damaged? 2. Is DNA fully replicated? Is DNA damage repaired? 3. Have spindle fibers formed? Have they attached to chromosomes correctly? 2. 1. 2001 Nobel Prize was awarded to 3 scientists who studied genes that regulate the cell cycle

29. CELL CYCLE CHECKPOINTS  MITOSIS ENTRY (G2/M)  Replication Complete  Growth/ Protein Synthesis adequate  No DNA Damage  S-PHASE ENTRY (G1/S)  Mitosis Complete ?signal - cyclin degradation  Growth/ Protein Synthesis (G1 CYCLINS)  No DNA Damage  OTHERS  MITOSIS EXIT: ?coupling to S-phase  S PHASE : coupling to mitosis ▪ also in response to DNA damage  G1 sequence of events ▪ signaling from cell surface

30.  Checkpoints are used by the cell to monitor and regulate the progress of the cell cycle.  Checkpoints prevent cell cycle progression at specific points, allowing verification of necessary phase processes and repair of DNA damage.  The cell cannot proceed to the next phase until checkpoint requirements have been met.  Several checkpoints are designed to ensure that damaged or incomplete DNA is not passed on to daughter cells.  Checkpoints are used by the cell to monitor and regulate the progress of the cell cycle.  Checkpoints prevent cell cycle progression at specific points, allowing verification of necessary phase processes and repair of DNA damage.  The cell cannot proceed to the next phase until checkpoint requirements have been met.  Several checkpoints are designed to ensure that damaged or incomplete DNA is not passed on to daughter cells.

31.  Two main checkpoints exist: the G 1 /S checkpoint and the G 2 /M checkpoint.G 1 /S checkpointG 2 /M checkpoint  G 1 /S transition is a rate-limiting step in the cell cycle and is also known as restriction point.  An alternative model of the cell cycle response to DNA damage has also been proposed; postreplication checkpoint.  p53 plays an important role in triggering the control mechanisms at both G 1 /S and G 2 /M checkpoints.  Two main checkpoints exist: the G 1 /S checkpoint and the G 2 /M checkpoint.G 1 /S checkpointG 2 /M checkpoint  G 1 /S transition is a rate-limiting step in the cell cycle and is also known as restriction point.  An alternative model of the cell cycle response to DNA damage has also been proposed; postreplication checkpoint.  p53 plays an important role in triggering the control mechanisms at both G 1 /S and G 2 /M checkpoints.

32. DNA Damage - Cell Cycle Arrest damage dependent checkpoints CELL No. DNA content asynchronous X-ray treated G1/S block G2/M block (6-9 hours) X-ray treated G1/S block G2/M block (6-9 hours) G1 - S - G2 wild-type loss of G1/S in p53 deficient cells G1 - S - G2

33. Spindles and Chromosomes more checkpoints!

34. CONCLUSIONS  CDKs, CYCLINs and regulators of Cyclin/cdks  Cell cycle events coupled to maintain the correct sequence  G0/G1, G1/S and exit from Mitosis: transitionsthat are often altered in cell transformation/oncogensis.  DNA Damage leads to rapid cessation of DNA synthesis, and cycle arrest at G1/S and G2/M  Stable state are G0, S exit, M exit  G2/M transition has greater impact on cell survival after DNA Damage, relative toG1/S

35. What are and what happens during the phases of the cell cycle? Which proteins are involved in the regulation of the cell cycle? Which cyclins and cyclin-dependent kinases are most important in individual phases of the cell cycle? What are four mechanisms for regulating cyclin-dependent kinase activity? What role do p53, p21, and pRb play in the G1 to S transition? What are and what happens during the phases of the cell cycle? Which proteins are involved in the regulation of the cell cycle? Which cyclins and cyclin-dependent kinases are most important in individual phases of the cell cycle? What are four mechanisms for regulating cyclin-dependent kinase activity? What role do p53, p21, and pRb play in the G1 to S transition?

36. http://highered.mcgrawhill.com/sites/st udent_/control_of_the_cell_cycle.html Please, Submit the online Quiz to my email; ggawish@ksu.edu.sa Animation & Quiz In your G0 phase you can play this fun animation, http://www.cell-action.com/cell_cycle/cell_cycle.html http://www.cell-action.com/cell_cycle/cell_cycle.html