Cell Cycle Checkpoints

How is MPF activity regulated?

1. Regulation of the concentration of mitotic cyclin → cell must synthesize mitotic cyclin to allow it to interact with Cdk before the cell can start M-phase

2. Regulation of MPF’s kinase activity by Wee1 (kinase) and cdc25 (phosphatase)

3. Kinase activity of cyclin-Cdk complexes can also be regulated by inhibitory proteins → S-phase cyclin-Cdk regulated by Sic1

How do Wee1 and cdc25 regulate mitotic cyclin-Cdk activity?

Mitotic cyclin and Cdk come together to form a complex; Cdk has two amino acids on it (threonine and tyrosine) that can become phosphorylated; when this complex first forms, it is not yet in an active state; Wee1 phosphorylates one of the amino acids, and this blocks the binding of ATP to the complex, so it is still inactive; another kinase, CAK, phosphorylates the Cdk again, and this increases the affinity of the Cdk for the substrate (makes it more specific), but the complex is still inactive; Cdc25 removes the phosphate from Cdk that is blocking ATP binding, this puts the cyclin-Cdk complex in an active state because ATP can now bind to it

What type of molecule is Wee1?

A kinase

What type of molecules is Cdc25?

A phosphatase

How does Wee1 activity relate to what Nurse found in the Wee fission yeast mutants?

Nurse found that Wee mutants underwent premature cell division, and the activity of the Wee1 kinase is to phosporylate mitotic cyclin-Cdk so that it is inactive; this makes sense because in the Wee mutants, Wee1 is defective, so mitotic cyclin-Cdk is not inhibited and the cells undergo cell division prematurely

What experiment showed how MPF activity is regulated?

Researchers used mutant S. pombe fission yeast cells; they found that in wild type cdc2 cells (have master kinase activity), if there was an excess of Wee1 or a deficit of Cdc25 (activity of cdc2 inhibited/not activated), the cells did not undergo division and instead just grew really long; they also found that in wild type cdc2 cells, if there was an excess of Cdc25 or a deficit of Wee1, the cells became Wee mutants and underwent cell division too early

What conditions will create big, long fission yeast mutants?

• no cdc2 activity, OR

• excess of Wee1, OR

• deficit of Cdc25

What conditions will create Wee fission yeast mutants?

• too much cdc2 activity, OR

• deficit of Wee1, OR

• excess of Cdc25

What effect does Wee1 have on mitotic cyclin-Cdk?

An inhibitory effect, it puts a brake on the activity of Cdk or else it would become active during G2-phase because that is when mitotic cyclin levels begin to rise

What effect does Cdc25 have on mitotic cyclin-Cdk?

A stimulatory effect, it takes the brake off of Cdk and allows it to become active during late G2 phase

How does the activation of MPF create a positive feedback loop?

Two of the substrates for MPF (mitotic cyclin-Cdk) are Wee1 and Cdc25; it phosphorylates Cdc25, which increases its activity, which is to activate MPF; MPF also phosphorylates Wee1, which inhibits Wee1 activity, which is normally to inhibit the activation of MPF

What is the result of the Wee1, Cdc25, MPF positive feedback loop on the cell cycle?

The positive feedback loop produces two stable steady states that the cell oscillates between (hysteresis), and this extends the range of cyclin concentration at which MPF activity is on/off; without the positive feedback loop, the activity of MPF would be very sensitive to small changes in cyclin concentration

What is hysterisis?

The term for when something exhibits switch-like behavior between two steady states

What is necessary for cell cycle oscillations?

There must be bi-stability of two steady states, and this requires fast positive feedback and slow negative feedback

What does f represent?

The ratio of active Cdc25 and Wee1 to inactive Cdc25 and Wee1; in order to get two stable steady states, you need the f ratio to be high (more active Cdc25 and Wee1 than inactive Cdc25 and Wee1)

What causes Cdc2 (MPF) activity to decrease once it reaches peak activity levels?

APC gets activated and it breaks down the cyclin, so Cdc2 can no longer be active

What is the negative feedback loop in cell cycle oscillation?

MPF activates APC, which inactivates MPF; this negative feedback loop is slower than the positive feedback loop of MPF with Cdc25 and Wee1

What is the primary way to shut off MPF activity?

By degrading mitotic cyclin via APC

What is a secondary way to “shut off” MPF activity?

By reversing the phosphorylations of the Cdk using a phosphatase (PP2A)

What is the function of Protein Phosphatase 2A (PP2A)?

It dephosphorylates the substrates that Cdc2/Cdc28 phosphorylates, counteracting its activity and allowing the cell to begin moving out of M-phase

What occurs when you create an S/G1 heterokaryon?

The G1 nucleus gets pulled forward into S-phase, accelerating the cell cycle; this suggested that there is a diffusible activating signal (S-phase promoting factor, SPF) that exists at or past the R-point

How is S-phase cyclin-Cdk activity regulated?

S-phase cyclin forms a complex with Cdk; this complex is not yet active because an inhibitory protein, Sic1, binds to the complex and blocks the kinase activity of the Cdk; Sic1 must be destroyed for S-phase cyclin-Cdk complex to become active; this occurs when G1/S-cyclin-Cdk (SPF) phosphorylates Sic1; phosphorylated Sic1 is recognized by a ubiquitin ligase (SCF), which polyubiquinates Sic1 and it is then broken down in a proteasome, allowing S-phase cyclin-Cdk to become active

What molecule is the SPF?

G1/S-phase cyclin-cdk; it phosphorylates Sic1, allowing S-phase cyclin-Cdk to become active so S-phase can begin

What happens when the cell senses DNA damage?

This triggers the synthesis of proteins that inhibit cyclin-Cdk complexes; there is a series of kinases that senses the damage in the DNA, and this leads to the phosphorylation of p53; phosphorylated p53 no longer interacts with its inhibitory protein, so p53 is now active; active p53 acts a transcription factor, promoting transcription of p21 gene; p21 acts like Sic1, inhibiting cyclin-Cdk complexes, specifically it inhibits G1/S cyclin-Cdk (SPF) by binding to it, and this blocks entry of cell into S-phase, giving the cell a chance to repair the DNA damage

What is a checkpoint in the cell cycle?

A point in the cell cycle at which progression arrests until the previous stage of growth/division has been completed properly or until certain conditions for cell division are met

When does a checkpoint get activated?

When conditions in the cell are not right; so if the checkpoint is active, then the cell cycle has stopped

What are the different checkpoints in the cell cycle?

• START/R-point checkpoint → right before entry into S-phase, checks if the DNA is intact, if cell has grown enough, and if there are the proper nutrients and growth factors present

• G2/M checkpoint → right before entry into M-phase, checks if all DNA was properly replicated

• Spindle-Assembly checkpoint → right before anaphase, checks if all chromosomes are properly attached to the mitotic spindle

• Spindle-Position checkpoint → right before telophase, checks if all chromosomes have properly segregated

How does the Spindle-Assembly checkpoint work?

At the kinetochore of a replicated chromosome, certainn Mad and Bub proteins bind and check if microtubules are properly attached to kinetochores, while this is happening, another population of Mad and Bub proteins receives a signal from the Mad and Bub proteins on the kinetochore; this signal tells the second population of Mad and Bub proteins to bind to a protein called Cdc20, which inhibits it from binding to APC (APC must be bound to Cdc20 to be active), so anaphase does not yet occur; once the kinetochore microtubules are properly attached, Mad and Bub are released from both the kinetochores and Cdc20, allowing APC to turn on and anaphase to occur

What is the spindle assembly checkpoint an example of?

It is an example of phase-dependency, meaning that the first event must be completed before the next event can start

If Mad and Bub are bound to Cdc20, what state is the spindle assembly checkpoint in?

The checkpoint would be on because APC activity is off, so the cell cycle is not yet proceeding

What occurs when you create a S/G2 heterokaryon?

The G2 nucleus remains in G2, it waits for the S nucleus to catch up before entering M-phase; it is also notable that the G2 nucleus does not re-enter S phase, suggesting that there is some block that prevents cells from returning to previous stages, and that DNA is only licensed to replicate once per cell cycle

What is the origin of replication?

This is where DNA replication begins; it is stretch of a few hundred base pairs rich in A’s and T’s → this is important because the A-T association only has two hydrogen bonds, whereas C-G association has three, so its easier to break the A-T association to separate the DNA strands; this site is also a recognition site for proteins involved in DNA replication

Is there only one origin of replication?

In eukaryotes there are multiple origins of replication; these origins activate in clusters simultaneously

Does an entire chromosome replicate at one time?

No, different regions of a chromosome replicate at different times depending on the level of DNA compaction; euchromatin tends to replicate earlier in S-phase than heterochromatin

What protein complex is present at the origin of replication?

The Origin Replication Complex (ORC); the ORC blocks replication during the cell cycle, so it must be displaced for replication to begin

What is the pre-replicative complex (pre-RC)?

It is a complex made of many proteins at the origin of replication, notably ORC and Helicase; during all phases other than S-phase, ORC is bound to Helicase and inhibits its activity

What happens to the pre-RC as the cell moves into S-phase? What about as it moves out of M-phase?

At the start of S-phase, S cyclin-Cdk becomes activated; active S cyclin-Cdk phosphorylates ORC, which causes it to no longer be bound to Helicase; Helicase gets phosphorylated (not by S cyclin-Cdk) and is now active and can begin unwinding the DNA, moving away from the origin; in late M-phase, the pre-RC reforms at the origins, and this is the license for DNA to be able to replicate again in the next cycle

What is the license for DNA to be able to replicate?

In order for DNA to be recognized as “competent” to replicate, the pre-RC must be present on the origins; after S-phase, the pre-RC is not present again until late M-phase, and this is why a cell in G2 will not be pulled back into S phase if it is mixed with an S-phase cell

What is the role of geminin?

It is a protein that affects how the ORC is associated to the DNA; it protects newly replicated DNA from pre-RC binding, so it is destroyed during late M-phase by APC to allow the pre-RC to bind to the DNA