The Cell Cycle

Question 1

What is the G0 phase of the cell cycle?

Answer 1

The G0 phase is a generally quiescent state that is found in non-proliferating cells common to multicellular eukaryotes. It can last for very long periods of time (indefinitely, as in the case of neurons) for fully differentiated cells. It comes off the G1 phase and can extend it for a long period of time.

Question 2

What is the primary function of the S phase of the cell cycle?

Answer 2

DNA replication.

Question 3

What type of experiment proves that DNA replication occurs at discrete 24 hour intervals?

Answer 3

A pulse/chase experiment using 3H-thymidine uptake as an indicator can be used to demonstrate that DNA replication occurs at 24 hour intervals in cultured HeLa cells.

Question 4

What happens when cells in S-phase are fused to G1 cells in a cell fusion study (using polyethylene glycol as a culture)?

Answer 4

The new cell will replicate both DNAs, indicating that the cytoplasm of the S-phase cell had an activator that influenced the G1 nucleus. In fusions involving multiple cells, if more S cells are used relative to G1, then the G1 nuclei enter DNA replication faster.

Question 5

What happens when an S-phase cell is fused to a G2 cell?

Answer 5

Only the S phase nucleus replicates while G2 (post S) remains refractory to S-activator. This insures that DNA is replicated only once because G2 is post S-phase and has already replicated its DNA. The G1 phase is sensitive to the S activator but the G2 phase is not.

Question 6

Once mitosis ends, what happens to MPF activity?

Answer 6

It quickly decays.

Question 7

What effect does MPF have on Xenopus Laevis oocytes?

Answer 7

Mitosis/maturation promoting factor (MPF) promotes the maturation of the oocytes and can act as a proxy for progesterone, which triggers the process of mitosis or meiosis.

Question 8

Describe the heterodimer structure of MPF.

Answer 8

MPF consists of two proteins: p34 (aka CDK) and p45 (aka cyclin). When complexed together in a heterodimer, these two proteins display a kinase activity. p34 is the catalytic subunit that catalyzes the phosphorylation of proteins on serines or threonines while p45 (cyclin) is the regulatory subunit.

Question 9

Describe the cycle of the MPF heterodimer complex.

Answer 9

p34 (CDK) remains constant and is influenced by the rise and fall of the p45 cyclins. In this way, MPF is really an allosteric enzyme complex. The heterodimer complex goes up and down in specificity, in what it is targeting.

Question 10

Why do serines and threonines become phosphorylated? (what do they have in common?)

Answer 10

CDKs (which exhibit kinase activity–kinases phosphorylate) target hydroxyl groups. Serines and threonines have these hydroxyl groups.

Question 11

At which two points does cell cycle regulation occur?

Answer 11

1. START (aka the restriction point). Once the cell passes this point, it will either complete the cell cycle or die.
2. G2-M transition. This is an important point for cell division control.

Question 12

What is the role of Cdh1?

Answer 12

Cdh1 controls the entry into anaphase and keeps the cell from leaving mitosis too soon.

Question 13

What is the role of G1 cyclin (CDK)?

Answer 13

1. Phosphorylation and inactivation of Cdh1.
2. Phosphorylation of retinoblastoma to activate transcription factors that start the expression of DNA replication enzymes and S-phase cyclins/CDKs.
3. At higher concentrations, phosphorylation and degradation of S-phase cyclin/CDK inhibitor via SCF proteosome ubiquitin pathway.

Question 14

What is the relationship between enzyme and high- and low-affinity targets?

Answer 14

High-affinity targets require less enzyme and low-affinity targets require the most enzyme and most cyclin kinase activity.

Question 15

What is the role of S-phase cyclin/CDK?

Answer 15

It phosphorylates and activates PRCs and thereby prevents them from rebinding to DNA.

Question 16

Describe the transition from G1 to S-phase.

Answer 16

In G1, ORC proteins bind to the origin, acting as a scaffold for licensing factors (MCMs) to also bind there. Then, when the activating factor–S-CDK–binds and initiates replication, S-phase can begin. Here, the replication fork begins to move.

Question 17

Once DNA is replicated, what happens to MCM proteins?

Answer 17

They are displaced and exported from the nucleus to ensure that there is no more DNA replication after the S-phase.

Question 18

What two factors affect the growth of S. pombe?

Answer 18

wee1 inhibits the growth of the yeast cell and Cdc25 activates the growth.

Question 19

What are the regulatory subunits of the kinase allosteric complex?

Answer 19

Cyclin B (aka Cdc13) and Cdc2.

Question 20

Wee1, a Cdc2 inhibitory kinase, has what target?

Answer 20

Wee1 targets the Y15 amino acid on Cdc2.

Question 21

What is the target of Nim1 kinase?

Answer 21

Nim1 inhibits wee1.

Question 22

What is the target of the Cdc2 activating kinase CAK?

Answer 22

The amino acid T161 on Cdc2. CAK catalyzes the phosphorylation of T161.

Question 23

What is the activation step of MPF?

Answer 23

Cdc25 encodes a phosphatase that is specific for Y15. Once activated, MPF further stimulates Cdc25 to produce more MPF and also inhibits wee1 to reduce MPF down-regulation. Together with the auto-phosphorylation of Cdc13, this results in an autocatalytic explosion of MPF activity that drives the cell into mitosis.

Question 24

What maintains the mitotic state and must be deactivated to get the cell out of mitosis?

Answer 24

Cdc13.

Question 25

Describe the phosphorylation cascade that causes cell cycle arrest in response to DNA damage in the G1 phase.

Answer 25

ATM detects DNA damage and then activates (via phosphorylation) Chk 2 checkpoint kinase to phosphorylate and stabilize the transcription factor p53. p53 turns on the synthesis of p21 mRNA that blocks the Cdk activity, which leads to cell cycle arrest.

Question 26

Describe the phosphorylation cascade that causes cell cycle arrest in response to DNA damage in the G2 phase.

Answer 26

ATR detects DNA damage and then activates (via phosphorylation) Chk 1 checkpoint kinase to phosphorylate Cdc25 (a phosphatase). Cdc25-P is bound by 14-3-3 adaptor and in this form is kept out of the nucleus so it is not present to activate Cdk, leading to cell cycle arrest.

Question 27

What does quiescent mean in terms of the cell cycle?

Answer 27

Many cells in the body are said to be quiescent, which means that they are in a state that will not lead them to an upcoming cell division, but they retain the capability to divide if conditions change.

Question 28

What must happen for a cell to leave the quiescent G0 phase and enter G1?

Answer 28

A cell must receive a growth-promoting signal to proceed from Go into G1 and thus reenter the cell cycle.

Question 29

What happens when a G1-phase cell and an M-phase cell are fused?

Answer 29

The chromatin of the G1-phase nucleus undergoes premature chromosomal compaction to form a set of elongated compacted chromosomes.

Question 30

What happens when a G2-phase cell and an M-phase cell are fused?

Answer 30

The G2 chromosomes are visibly doubled, reflecting the fact that replication had already occurred.

Question 31

What happens when an S-phase cell and an M-phase cell are fused?

Answer 31

The S-phase chromatin becomes compacted, but because replicating DNA is especially sensitive to damage, the compaction in the S-phase nucleus led to the formation of “pulverized” chromosomal fragments rather than intact, compacted chromosomes.

Question 32

What findings did the cell-fusion experiments reveal?

Answer 32

The cytoplasm of a mitotic cell contains diffusible factors that can induce mitosis in a nonmitotic (ie interphase) cell. This suggests that the transition from G2 to M is under positive control; that is, the transition is induced by the presence of some stimulatory agent.

Question 33

What did experiments on oocytes reveal about the role of enzymes in initiating mitosis?

Answer 33

1. Progression of cells into mitosis depends on an enzyme whose sole activity is to phosphorylate other proteins.
2. The activity of this enzyme is controlled by a subunit whose concentration varies from one stage of the cell cycle to another.

Question 34

Which kinase controls the two regulation points of the cell cycle, and which two differing cyclins does it bind to at each point?

Answer 34

cdc2 kinase controls the regulation points from G1->S (START) and from G2->M. At the transition from G1->S, the cdc2 kinase binds with G1/S cyclins, and at the transition from G2-> M, the cdc2 kinase binds with mitotic cyclins.

Question 35

What controls the exit from mitosis and entry to G1?

Answer 35

A rapid decrease in Cdk activity that results from a plunge in the concentration of the mitotic cyclins.

Question 36

What effect does the protein kinase Wee1 have on the Cdk enzyme?

Answer 36

Wee1 phosphorylates a tyrosine residue on CDK, making the enzyme inactive (this overrides any activity by CAK). Cells with a mutant wee1 gene cannot maintain the CDK in an inactive state and divide at an early stage in the cell cycle, producing smaller cells.

Question 37

What effect does the protein kinase CAK have on the Cdk enzyme?

Answer 37

CAK phosphorylates a threonine residue on Cdk, which is a step towards activating the Cdk.

Question 38

What switches Cdk into its active state?

Answer 38

At the end of G2, the inhibitory phosphate at tyrosine 15 is removed by a phosphatase named Cdc25. Removal of this phosphate switches the stored cyclin-Cdk molecules into the active state, allowing it phosphorylate key substrates and drive the cell into mitosis.

Question 39

What happens to the phenotype of cells that have a mutant cdc25 gene?

Answer 39

These mutants cannot remove the inhibitory phosphate from the Cdk and cannot enter mitosis.

Question 40

What is the function of the complexes SCF and APC?

Answer 40

These two classes of multisubunit complexes function as ubiquitin ligases that regulate the cell cycle. They recognize proteins to be degraded and link those proteins to a polyubiquitin chain, which ensures their destruction in a proteasome.

Question 41

When is the SCF complex active?

Answer 41

The SCF complex is active from late G1 through early mitosis and mediates the destruction of G1/S cyclins, Cdk inhibitors, and other cell cycle proteins.

Question 42

What happens if there are mutations that inhibit SCF complexes?

Answer 42

This inhibits SCFs from mediating the proteolysis of key proteins, which prevents the cell from entering S phase and replicating its DNA.

Question 43

What does the APC complex degrade?

Answer 43

The APC complex acts in mitosis and degrades a number of key mitotic proteins, including the mitotic cyclins. Destruction of the mitotic cyclins allows a cell to exit mitosis and enter a new cell cycle.

Question 44

Define checkpoints.

Answer 44

Checkpoints are surveillance mechanisms that halt the progress of the cell cycle if 1. any of the chromosomal DNA is damaged, or 2. certain critical processes, such as DNA replication during S phase or chromosome alignment during M phase, have not been properly completed.