L7

Question 1

What is a cell cycle checkpoint?

Answer 1

A point in the cell division cycle which leads to the next cell cycle transition if the necessary cell cycle events have been completed

Question 2

What are the types of feedback controls?

Answer 2

Sensing mechanism for the completion of the event

Output signal from the sensor

A response element that controls the cell division cycle

Question 3

What 2 classes of mutation were expected as a response of cells to DNA damage?

Answer 3

One class involved in repairing damage

One class involved in cell cycle arrest

Question 4

What happens if you put DNA damage into cells in G2?

Answer 4

The cell cycle arrests

Question 5

What happens if you add caffeine to cells with DNA damage in G2?

Answer 5

Cells no longer arrest

Here the caffeine prevented the cell signal checkpoint from detecting the response of DNA damage so it went through mitosis

Caffeine has so many affects its hard to tell exactly what its doing to cause this

Question 6

What evidence did Hartwell get of the cell signal checkpoint acting in G2 in cerevisiae?

Answer 6

When the wildtype grow, it divides, buds and forms a little microcolony

He took WT yeast cells and irradiated them when in G2 phase (have passed S phase) & found that unlike the WT that were untreated, they stopped dividing, they continued metabolizing but the cell cycle stopped – after a period of time the cells moved off again

Question 7

How can we detect the affect of mutations on the cell signalling checkpoint in G2?

Answer 7

Looked for mutations that were sensitive to radiation and analysed them under a microscope to see how they responded

If the mutations affected the repair mechanism, then the cell should arrest just fine

If the mutations affected the cell cycle mechanism, the repair mechanism would be unaffected, but cells would arrest

Question 8

Affect of a Rad52- mutant on the cell signalling checkpoint in G2?

Answer 8

Rad52- mutant when irradiated in G1 arrests like the WT cell – has no repair mechanism

Rad52- mutant blocked repair but not the cell cycle arrest – eventually died as wasn’t repaired

^ repair mechanism mutation

Question 9

Affect of a Rad9- mutant on the cell signalling checkpoint in G2?

Answer 9

Doesn’t behave the same way as the Rad52- mutant

When these cells were treated in G2 they didn’t arrest & continued dividing even though they were damaged – damage keeps growing

Accumulation of microcolony of dead cells – checkpoint mutation

Question 10

Checkpoints that block in G2 in S. cerevisiae

Answer 10

DNA damage checkpoint: Rad9, Mec1, Mec2, Mec3
• Either DNA replication, cdc mutations or radiation damage

Unreplicated DNA checkpoint: Mec1, Mec2
• Hydroxyurea can be used to block DNA replication (inhibits ribonucleotide reductase)

Question 11

Checkpoints that block G2 in S. pombe

Answer 11

DNA damage checkpoint: Rad1, Rad24

Unreplicated DNA checkpoint: Rad1

Question 12

Why is it called Rad?

Answer 12

Because its sensitive to radiation

Question 13

What happens when you get DNA damage in S. cerevisiae?

Answer 13

You usually block G2

Question 14

What happens when you lack rad9 or mec3 in S. cerevisiae?

Answer 14

You block the inhibition of the G2 phase from damaged DNA

The cells undergo mitosis when damaged

Question 15

What happens when you lack mec1 or mec2 in S. cerevisiae?

Answer 15

The cell no longer arrests & can enter mitosis with either damaged DNA or unreplicated DNA

Question 16

What does Rad24 do in S. pombe?

Answer 16

Rad24 affects the response to DNA damage in G2

Question 17

What does Rad1 do in S. pombe?

Answer 17

Rad1 responds to both DNA damage & unreplicated DNA

Question 18

What does cdc2-F15 do in S. pombe?

Answer 18

Important in the unreplicated DNA pathway

Cdc2-F15 mutation causes pombe to undergo division at a very small size
– Cells that have not replicated their DNA with that mutation with undergo mitosis regardless
– Sensing mechanism is no longer working = disaster for the cell

Question 19

Mechanism of cell cycle arrest of cdc20 in S. cerevisiae

Answer 19

Experiments suggest Cdc20 is important for DNA damage pathway but not the unreplicated DNA pathway

Cdc20 is also very important in the spindle pole checkpoint

Question 20

Mechanism of cell cycle arrest of cdc28 in S. cerevisiae

Answer 20

Mutation of Tyr19 of Cdc28 to the ‘active’ form has no affect in the cell cycle arrest caused by DNA damage or the presence of unreplicated DNA

Question 21

What 2 classes of mutation were expected as a response of cells to mitotic spindle defects?

Answer 21

One class involved in spindle structure

One class involved in cell cycle arrest

Question 22

Checkpoint & feedback controls in mitosis: C. cerevisiae

Answer 22

Mad1, Mad2, Mad3 (mitotic arrest deficient)

Bub1, Bub2, Bub3 (budding uninhibited by benzimidazole)

All 6 proteins are involved in the checkpoint

Question 23

Mad/Bub dependent control in S. cerevisiae

Answer 23

1. Drugs that depolymerise the spindle
2. Mutations that affect spindle function
3. Multiple copies of a plasmid containing a centromere

Question 24

What are kinetochores?

Answer 24

The centromere & the proteins attaching it to the mitotic spindle

If you put lots of extra centromeres into a cell, it activates the checkpoint – cell gets confused about what’s happening

Mitotic checkpoint is probably monitoring defective kinetochore structure

Question 25

Experiments done on cerevisiae cells

Answer 25

Has a single copy of a plasmid, it divides and forms a colony

If there’s multiple plasmids with a centromere (cerevisiae has 16 chromosomes so has 16 centromeres) – involving other ones produces a problem as there’s only so much space for centromeres on the mitotic spindle
– Leads to the cell cycle being delayed as there’s competition between plasmids – it then fires ahead after everything is attached properly

If there’s a mad- mutant there is no delay
– This leads to chromosome loss as chromosomes are not being attached properly before it goes through the next cycle

Question 26

What happens in mitosis?

Answer 26

Everything lines up on the metaphase plate & is then pulled apart to opposite poles when ready

Question 27

How was the mitosis checkpoint originally thought to be linked to cyclin proteolysis?

Answer 27

As cells exit mitosis G2 cyclins get degraded - thought to be the key element to drive the process

Question 28

What happens when you add benomyl to cells in M phase?

Answer 28

Causes depolymerization of the spindle, results in the chromosomes not attaching properly

The cell cycle checkpoint Mad and Bub is activated to block cyclin proteolysis – this blocks the step from the cell moving into anaphase until the cell is ready

Once aligned, cyclins can be degraded, and chromosomes separated

Question 29

What happens when you add a mad- mutant to cells in M phase that are treated with benomyl?

Answer 29

When you introduce a mad- mutant you lose the feedback control mechanism

Cyclin proteolysis occurs too early & the cells separate too early leading to chromosome loss & cell death

Question 30

What do Mad1/2/3 do in the cell?

Answer 30

Genetics & biochemistry indicate that Mad2 & Mad3 & possibly Mad1 interact with Cdc20 & block its function when the mitotic checkpoint is activated

Mad1 phosphorylation is increased at the checkpoint

Mad2 is not an isoprenyl transferase

Question 31

What is actually going on in the cell in M phase?

Answer 31

When the chromosomes are lined up on the pole on the metaphase plate, they are stuck together by a protein called cohesin which puts it under tension because the spindle wants to pull to either pole but can’t do this

Normally in mitosis, when its ready to go, seperase degrades the glue (cohesion)

Seperase is inhibited by sercurin – prevents the enzyme degrading the glue

Sercurin is proteolysed in metaphase

This is what the checkpoint depends on

When you want to stop the process, you don’t let securin be degraded

Question 32

Mutants in S. cerevisiae at the M phase

Answer 32

Mad1 is phosphorylated to activate the checkpoint

If you make mad2, bub1 and bub3 triple mutant you don’t get phosphorylation of Mad1 – shows these proteins are leading to the phosphorylation event

But if you make mutations in mad3 and bub2, phosphorylation occurs

Question 33

Checkpoint & feedback controls in mitosis: C. pombe

Answer 33

Mad2 protein has been identified that regulates mitosis

Mad2 interacts & regulates the activity of Slp1, a Cdc20 homologue, which also targets proteins for degradation

Question 34

Checkpoints, feedback controls & timings of the cell cycle events

Answer 34

1. Substrate/product relationship where product of an early event is a substrate for a later event
2. Timing – both processes initiate together but one takes longer to complete
   – Eg. single strand DNA at DNA replication is “repaired” before mitosis occurs
3. Feedback control – requires detection, signal by the incomplete event leading to a biochemical reaction