Topic D1

Question 1

What are the phases of the cell cycle?

Answer 1

• G1 phase: the cell has come out of mitosis and must grow, synthesise its mRNA and proteins to carry out its function and provide the means for DNA replication (1/3 of cell cycle)
  -Synthesis (S) phase: replication of the DNA occurs (~1/2 of cell cycle)
  -G2 phase: pre-mitotic phase in which there is rapid cell growth and protein synthesis and the cell “gears up” for mitosis (~1/6 of cell cycle)
  M phase:
• Mitosis: nuclear division of the cell divided into prophase, metaphase, anaphase and telophase
• Cytokinesis: cytoplasmic division of the two replicated cells
• G0: a cell cycle arrest phase in which the cell ceases the replicative cycle; some cells are permanently arrested in this phase, others can move into the cell cycle once more based on signals. Most cells spend the majority of their lifespan in the G0 phase

Question 2

What is prophase?

Answer 2

• Step 1 of mitosis:
  Early Prophase:
• DNA begins to condense into chromosomes
• Mitotic spindle (structure of microtubules) begins to form
• The nucleol(us/i) degrade

Late Prophase/Prometaphase:

• The chromosomes finish compacting are very compact
• The nuclear envelope breaks down, releasing the chromosomes
• The mitotic spindle grows more and some of these microtubules (called kinetochore microtubules) attach to a patch of protein on the centromere of each sister chromatid called the kinetochore
• Some microtubules extend from each centromere to the edge of the cell forming the aster

Question 3

What is metaphase?

Answer 3

• The spindle fibres have captured all of the chromosomes and has lined them up in the middle of the cell at the metaphase plate
• The two kinetochores of each sister chromosome are attached to microtubules from opposite spindle poles
• Spindle assembly checkpoint: the cell will check that all the chromosomes are at the metaphase plate with their kinetochores correctly attached to microtubules before the cell will move into anaphase

Question 4

Anaphase:

Answer 4

• The sister chromatids separate from each other are pulled towards opposite ends of the cell
• Microtubules from each pole push against each other and elongate the cell
• An actin myosin ring forms in the middle of the cell

Question 5

Telophase:

Answer 5

• The mitotic spindle is broken down
• Two new nuclei form
• The chromosomes begin to de-condense
• The actin myosin ring pinches and creates a cleavage furrow

Question 6

What are the 2 mechanisms by which proteins needed for mitotic events are “switched on/off” during different phases of mitotis?

Answer 6

1. Phosphorylation:
   - When proteins with hydroxyl groups on Serine or Threonine residues are phosphorylated by protein phosphatase or dephosphorylated by protein kinases it changes their structure (overall charge and shape) and can make them into an active or inactive form.
   - Generally achieved via the activity of a family of enzymes: cyclin dependent kinases (CDKs)
2. Degradation:
   the process of ubiquitin-proteasome mediated protein degradation

Question 7

How does the process of ubiquitination of a target protein occur?

Answer 7

1. Adding the polyubiquitin chain to the target protein:
   - An activated E1 enzyme has a ubiquitin molecule added to it via its –SH group
   - E1 and the bound ubiquitin molecule bind to E2 and E3.The E2 and E3 enzymes form a complex called ubiquitin ligase. E2 is the part of the complex that accepts the ubiquitin molecule with its –SH group and transfers the ubiquitin onto the target protein and E3 is the part of the complex that recognises and binds the target protein
   - Ubiquitin attached to E1 is transferred to ubiquitin ligase (E2+E3 complex) and E1 is discarded
   - Ubiquitin ligase will bind the target protein which is displaying a degradation signal (such as poor folding or merely being a present substrate for an active E3) via E3 and E2 will transfer the ubiquitin molecule onto the amino group of a lysine side chain on the target protein
   - E1 follows this initial ubiquination of the target protein by ubiquitin ligase by adding more ubiquitin molecules creating a multiubiquitin chain on the target protein

Question 8

How does the proteosome degrade the ubiquinated protein/

Answer 8

• The proteosome is an ATP dependent protease
• Only proteins with a polyubiquitin chain attatched can enter the active site in which the protease activity takes place and the peptide is degraded the amino acids are recycled.

Question 9

Why are CDKs despite always being present in the cell only active during certain periods of the cell cycle?

Answer 9

• These Cdks are only active during certain periods of the cell cycle because they must bind to another protein called cyclin in order to function. Cyclin proteins are only synthesised at certain points in the cell cycle; therefore despite the constant presence of Cdks they will only be active when their specific cyclin has been synthesised and is present.
  e. g. G1/S-cyclin is only synthesised during the G1 phase and therefore the G1/S-Cdk is active at this time

Question 10

How does cyclin activate CDK?

Answer 10

1. Binding of the cyclin molecule causes a confirmation change in Cdk meaning the T loop of inactive Cdk moves out and opens the ATP pocket making it available to be used to phosphorylate serine/threonine residues in proteins thus making the Cdk partly active
2. The shift in the T loop due to cyclin binding means that a Cdk-activating kinase (CAK) is able to phosphorylate the T loop which makes it bind substrate more readily and strongly thus increasing the Cdks activity

Question 11

How does Mitotic Cyclin- CDK (M-CDK) drive mitosis?

Answer 11

M-CDK drives:
1. Degradation of the nuclear membrane by phosphorylating nuclear lamin proteins and nuclear pore complexes

2. Facilitating the condensatation of DNA into chromosomes- because M-CDK degrades the nuclear membrane the cytosolic protein condensin is able to contact DNA and induce its condensation into chromatins
3. Assists in the formation of mitotic spindle poles and activation of motor proteins- M-CDK phosphorylated micro-tubule associated proteins (MAPs) so they are no longer able to function and stabilise interphase microtubules. M-CDK phosphorylates and activates motor proteins.
4. Activates anaphase promoting complex

Question 12

What is the function of anaphase promoting complex (APC)?

Answer 12

• Anaphase promoting complex has ubiquitin ligase activity which unbiquitilates the protein securin (which is bound to inactive seperase) causing it to be degraded by the proteasome
• Degradation of securin frees and actives seperase which cleaves the cohesin molecules holding sister chromatids togethers driving the division of chromosomes.
• The APC also inactivates M-Cdk

Question 13

How does the anaphase promoting complex inactivate M-CDK at the end of metaphase?

Answer 13

• APC has ubiquitin ligase activity and ubiquitylates M-cyclin at the end of metaphase
• This ubiquitylation causes the M-cyclin molecule to be degraded by the proteosome
• Without M-cyclin bound to M-CDK; M-CDK is no longer active and all the proteins it phosphorylated can be desphorylated by phosphatases allowing the new cells to reform their nuclear membranes and interphase microtubule structures.