1. Agenda 2/26
DNA Replication Review
Cell Cycle Notes
Board Games
Homework: Cell Cycle ‘Click and Learn’, Turn in: Chargaff WS answers
Warm Up: Write the steps of DNA replication

2. Steps of DNA Replication
1. Helicase ‘unzips’ hydrogen bonds between base pairs separating the two strands of DNA. Topoisomerase is ahead of helicase to relieve tension in DNA strand
2. Primase lays down RNA bases as a primer to start DNA replication
3. DNA polymerase III begins at 3’ end of the template strand. This creates one leading strand and one lagging strand with okazaki fragments.
4. DNA polymerase I replaces original RNA nucleotides with DNA nucleotides
5. Ligase glues okazaki fragments together to create a continuous strand

3. If DNA replication doesn’t occur or doesn’t occur correctly, what do we want the cell to do??

4. What is the different between normal cells and cancerous cells?
Cancer Cell Animation

5. Cells & Cell Reproduction
The "life of a cell" is termed the cell cycle.
The cell cycle has distinct phases, which are called G1, S, G2, and M.
Cells that have temporarily or reversibly stopped dividing are said to have entered a state of quiescence called G0 phase.
Cells that cannot reproduce are destined to die and are called “terminal” cells (red blood cells, nerve cells, muscles cells, etc.). They remain in the G1 phase or G0 phase. Cardiac muscle cells stop in G2 and therefore have twice the normal amount of DNA.
Note: “G” stands for gap although some books refer to it as growth.

6. Steps in the Cell Cycle G1: Growth, longest phase (10 hours)
S: Synthesize DNA/DNA replication, additional organelles are made (4 hours)
G2: More growth, making proteins needed for mitosis (4 hours)
M: Mitosis and Cell Division

7. The Amount of DNA Varies During the Cell Cycle
Ask students to interpret the graph emphasizing the amount of DNA throughout each segment of the cell cycle. Emphasize the changes and more importantly, the CAUSE of the changes. Don’t neglect the significance of the dramatic decrease in DNA at the very end of the M phase. Emphasize the return to the “1 X” level.
This graph represents the amount of DNA found in the cell during the cell cycle.

8. Cell Cycle Checkpoints

9. How do the Cell Checkpoints Actually Work?
Cyclins and Cyclin Kinases
Proteins like p53

10. Internal Controls of the Cell Cycle
The control of the cell cycle is dependent on an accumulation of “signal molecules”.

11. Control of the Cell Cycle
Cyclins are proteins that accumulate to a certain tipping point that can cause the cell to advance to the next stage in the cycle
Cyclin-Dependent Kinase (CDK)- A chemical (enzyme) that helps speed up actions in a cell
Cyclins were originally named because their concentration varies in a cyclical fashion during the cell cycle. By definition, a CDK binds a regulatory protein called a cyclin. Without cyclin, CDK has little kinase activity; only the cyclin-CDK complex is an active kinase.
One of the largest groups of kinases are protein kinases, which act on and modify the activity of specific proteins. Kinases are used extensively to transmit signals and control complex processes in cells. More than five hundred different kinases have been identified in humans. Their enormous diversity, as well as their role in signaling, makes them an object of study.

13. Cyclins vs. Kinases
Certain cyclins are made at certain times during the cell cycle, and their concentration will rise and fall. Cyclins are also destroyed after they are no longer needed by the cell.
CDKs are not destroyed as they are only activated or deactivated.
Ask students to interpret this graph. Specifically ask which cyclin affects which phase of the cell cycle.

14. Kinases Phosphorylate Cell Signal Molecules
Kinases are a type of enzyme that transfers phosphate groups from high-energy donor molecules, such as ATP, to specific substrates, a process referred to as phosphorylation.
Note: Kinases are not to be confused with phosphorylases, which carry out phosphorolysis, the breaking of a bond using an inorganic phosphate group; or with phosphatases, which remove phosphate groups. (They all start to sound alike, don’t they?)

15. Cyclins Activate Kinases
Once the cyclin-CDK complex is formed, they can directly act on the cell
Ex: MPF (mitosis promoting factor) can help break apart the nucleus and build the spindle fibers in preparation for division
The cyclin attaches to the CDK. It is now called a cyclin-CDK complex. The complex that regulates the M (mitosis) portion of the cell cycle has 3 names (ugh!): the maturation-promoting factor, mitosis-promoting factor or M-Phase promoting factor. Luckily they are all referred to as “MPF”.
MPF is activated at the end of G2 by a phosphatase, which removes an inhibitory phosphate group added earlier.
The MPF is also called the M phase kinase because of its ability to phosphorylate target proteins at a specific point in the cell cycle and thus control their ability to function.
Here’s an example of how it initiates mitosis:
MPF promotes the entrance into mitosis (the M phase) from the G2 phase by phosphorylating multiple proteins needed during mitosis. The steps follow:
The nuclear lamina depolymerizes causing it to disassemble which in turn causes the nuclear membrane to disassemble
Histone H1 binds to the DNA in chromosomes, causing the chromosomes to condense
Cytoskeletal proteins allow cytoskeletal filaments to assemble which leads to:
Formation of the mitotic spindle which separates the daughter chromosomes
formation of the cleavage furrow by microfilaments which allows cytokinesis (constricting the cell at the center) to occur resulting in the formation of two new cells

16. Cyclins Activate Kinases
Once the CDK complex does its job the cyclin is destroyed.
In the cell, the concentration of cyclins will rise and fall depending on the phase of the cell cycle.
When the cyclin is destroyed the CDK returns to an inactive form.
Emphasize that CDKs are not destroyed in the cell cycle. It is only the cyclins that are destroyed and this destruction deactivates the CDKs.
Note- Many cyclins are destroyed by first being tagged with small proteins called ubiquitin. Once tagged, large protein complexes called proteasomes, recognize the tagged cyclins and sequester the cyclins. The cyclins are then degraded into smaller, nonfunctional polypeptide chains and released to recycle the amino acids. This material will be covered again in gene regulation.

17. Cyclins Activate Kinases
Remind the students that the kinases are phosphorylating one or more chemical signals needed for the next phase of the cell cycle to occur. In this, the M cyclin/CDK complex or MPF triggers the actual start to mitosis. Notice that there is a critical amount of M cyclin concentration needed in order for to provide enough MPF activity to cause mitosis to begin.
Above is an example of how the M cyclin concentration affects MPF or M/CDK activity.

18. Cyclic Nature of Cyclins in the Cell Cycle
Revisit this graph. Emphasize again that a number of cyclins are involved in the cell cycle and that they activate a number of different kinases.
The term cyclin was coined by R. Tim Hunt who discovered them while studying the cell cycle of sea urchins cells. He said he named after his hobby of cycling and at the time he did not realize the role of these molecules in the cell cycle. However due the cyclic nature of the concentration of these compounds and their role in the cell cycle, the name stuck. Cyclins (D, E, A, B) are named based on the their protein structure and conserved parts. Older classification of cyclins is based on their role in the cell cycle. Most introductory books use the terms like S cyclin and M cyclin.
R. Tim Hunt along with Leland Hartwell and Sir Paul Nurse received the Nobel Prize in Medicine in 2001 for their discovery and research in the role of cyclins and CDKs in the cell cycle.
This graph displays the cyclic nature of various cyclins in a given cell cycle

19. Function of Cyclin/CDK Complex
Different Types of Cyclins
Cyclin/CDK Complex
Cyclin
Function of Cyclin/CDK Complex
G1-CDK
Cyclin D
Drives the transition G1  S transition
G1/S-CDK
Cyclin E
Cyclins bind to CDK at the end of G1 and commits the cell to DNA replication.
S-CDK
Cyclin A
Cyclins bind the CDK during S and are necessary for the initiation of DNA replication
M-CDK
Cyclin B
Cyclins promote the events of Mitosis
Students are not required to know the names cyclin/CDK complexes but S and M cyclin/CDK are illustrative examples.
These are some known cyclin/CDK complexes and their role in the cell cycle.

20. Cyclins/CDKs Control the Cell Cycle
The cell cycle has a number of several external and internal checkpoints much like a timer or clock. Often, moving past these check points involves chemical signals that have been phosphorylated by cyclin-CDK complexes.
G1/S (R point) checkpoint is the primary determining factor for cell division to take place. Growth factors are affecting the cell cycle, and cells are growing. Once the R point is passed the DNA is going to be replicated. If a cell receives a go-ahead signal at this check-point, it will complete the cell cycle and divide. However, if the cell does not receive the go-ahead signal in G1, the switches to a nondividing state called G0. If the cell passes the G1 checkpoint, it is usually committed to cell reproduction.
This checkpoint represents the commitment for starting the process of mitosis. This checkpoint also ensures that the DNA has been replicated correctly. If the DNA has been damaged, then the cell does not continue to mitosis. Once the CDK and cyclin combine, it is called “maturation promoting factor" or “mitosis promoting factor” or MPF.
3. This checkpoint makes sure that chromatids separate and ultimately move to opposite poles.
The M/ spindle checkpoint ensures that all the chromosomes are attached to the spindle in preparation of mitosis. The separation of the chromatids is irreversible.
Once chromatids are replicated they are held together by a protein substance called cohesin protein. Another protein called seperase can destroy this protein but has two forms active and inactive.
Securin is a protein that can activate seperase. Securin is activated by anaphase promoting complex or APC. Note-This is not a cyclin/CDK complex.
In many cells this occurs during anaphase, however in vertebrates, all of the cohesin is removed during chromatid condensation except the cohesin at the centromeres. Once the cohesin is completely removed, then the tension of the microtubules causes the separation of the chromatids.
APC also destroys M and S cyclins in order to drive the cell out of mitosis. APC can attach ubiquitin to the M and S cyclins tagging them for destruction by proteasomes.

21. P53
A protein that inhibits various cyclins to STOP the cell cycle if DNA damage occurs.
Why do we want this if DNA damage occurs?

22. P53

23. P53 and HPV

24. External Factors Also Influence the Cell Cycle
Cell reproduction and the healing of a wound.
Hemostasis
Inflammation
Proliferation
Remodeling
External Signals also affect cell division. Mammalian cells need certain nutrients and regulatory proteins. In addition external growth factors are can determine cell division in mammals. For example, when the skin has been damage (wound), platelets release a substance called platelet-derived growth factor (PDGF). This growth factor stimulate fibroblasts cells to start to reproduce and make scar tissue.
Hemostasis-Bleeding is contained via blood clotting.
Inflammation-Bacteria and debris removed. Damaged platelets release platelet derived growth factors. These external growth factors signal fibroblast to start to reproduce and make new cells.
Proliferation- Fibroblast reproduce forming an extra cellular matrix
Remodeling- Extra collagen and cells that are not needed are removed.
Graphic