Chapter 17

Question 1

Why are there gaps between the S phase and the M phase?

Answer 1

The gaps between S phase and M phase are required partly to permit cells the time they need to grow and double their mass of proteins and organelles. The two gaps also provide time for the cell to monitor the internal and external environment to ensure that conditions are suitable and preparations are complete before beginning S phase and mitosis

Question 2

What is Cyclin-dependent kinase (Cdk)?

Answer 2

A member of the family of protein kinases that have to be complexed with a cyclin protein in order to act.

Question 3

What is Anaphase-promoting complex or cyclosome (APC/C)?

Answer 3

The ubiquitin ligase that promotes the destruction of a specific set of proteins, thereby promoting the separation of sister chromatids and the completion of M phase

Question 4

What is M-Cdk?

Answer 4

The cyclin-Cdk complex responsible for stimulating entry into mitosis at the G2-M checkpoint

Question 5

What is Cyclin?

Answer 5

A family of proteins that rise and fall in concentration in step with the eukaryotic cell cycle, thereby regulating the activity of the crucial protein kinases that control progression through the cell cycle

Question 6

What is the Metaphase to anaphase transition?

Answer 6

The checkpoint in the cell cycle, where the control system stimulates sister-chromatid separation, leading to the completion of mitosis and cytokinesis

Question 7

What is Cdc25?

Answer 7

A phosphatase which important for the activation of the M-Cdk complex

Question 8

What is APC/C?

Answer 8

Anaphase promoting complex

Question 9

What is Cdc20?

Answer 9

It is required for two microtubule-dependent processes, nuclear movement prior to anaphase and chromosome separation

Question 10

What is Cdh1?

Answer 10

The CDH1 gene provides instructions for making a protein called epithelial cadherin (E-cadherin)

Question 11

What is CAK?

Answer 11

CDK activating kinase

Part of the second step in activation of Cdk by phosphorylating threonine residue 160 in the CDK activation loop

Question 12

What is CKI?

Answer 12

Cyclin-dependent kinase inhibitor protein?

function as tumor suppressor proteins. 
Are involved in cell cycle arrest and G1 phase

Question 13

What is SCF?

Answer 13

SCF complexes are E3 ubiquitin ligases

SCF controls the transitions between G1/S and G2/M phase

Question 14

What is M-cyclin?

Answer 14

The activation of M-Cdk begins with the accumulation of M-cyclin, whereafter Cdk1 associates with M-cyclin, resulting in M-Cdk complex

Question 15

What is G1-Cdk?

Answer 15

Complex formed in vertebrate cells by a G1-cyclin and the corresponding cyclin-dependent kinase (Cdk)

Question 16

What is G1/S-Cdk?

Answer 16

Complex formed in vertebrate cells by a G1/S-cyclin and the corresponding cyclin-dependent kinase (Cdk)

Question 17

What is S-Cdk?

Answer 17

Complex formed in vertebrate cells by an S-cyclin and the corresponding cyclin-dependent kinase (Cdk).

Question 18

What is G2-M transition?

Answer 18

The G2/M transition is a decisive point in a cell’s life cycle. The point at which, after successfully completing a second growth phase (G2 phase) following the replication of its DNA (S phase), it begins mitosis (the phase during which it physically separates itself into two daughter cells)

Question 19

What is Wee1?

Answer 19

Wee1 is a nuclear kinase belonging to the Ser/Thr family of protein kinases in yeast

It is a key regulator of cell cycle progression. It influences cell size by inhibiting the entry into mitosis, through inhibiting Cdk1

Question 20

How are the levels of cyclins regulated in comparison to the levels of Cdks during the cell cycle?

Answer 20

The concentrations of the cyclins rise and fall during the cell cycle whereas the concentration of the Cdks is constant throughout the cell cycle. They are regulated by transcription and by ubiquitin-mediated degradation

Question 21

What mechanisms are involved in the regulation of cyclin-cdk complexes?

Answer 21

Regulated by phosphorylation and de-phosphorylation, and by the binding of Cdk inhibitor proteins (CKIs) and rates of synthesis and proteolysis of the cyclin subunits

Question 22

How are the sister chromatids held together along their length until they separate at mitosis?

Answer 22

Cohesin complexes are deposited along the length of the sister chromatids during DNA replication in S phase.

Intertwining of DNA strings contributes to the cohesion.

The enzyme topoisomerase II gradually disentangles the sister chromatids between S phase and early M phase.

Dissociation of cohesion complexes from sister chromatids are also regulated by PLK1-mediated phosphorylation and removed enzymatically by separase.

Question 23

What positive feedback mechanisms are involved in stimulating the M-Cdk complexes?

Answer 23

The activated M-Cdk complex stimulates the activity of the Cdc25 phosphatase, inhibits the activity of the Wee1 kinase, and stimulates expression of the M-cyclin subunit.

Question 24

What events in the M-phase are stimulated by the M-Cdk complex?

Answer 24

Assembly of the mitotic spindle, bipolar attachment of the sister chromatids, chromosome condensation (by phosphorylation of condensin), nuclear envelope breakdown, and rearrangement of the cytoskeleton and Golgi apparatus.

Question 25

What is the structural basis of the activation of Cdk?

Answer 25

Binding between the (S- in fig. 17-12)cyclin and Cdk leads to conformation change and unblocking of the active site by a T-loop in Cdk. Full activation of the complex further requires phosphorylation of the Cdk by a Cdk activating kinase (CAK) at a threonine residue in the T-loop. This improves the ability of the active site to bind to its substrates.

Question 26

How many kinetochores are there in a human cell in the early M-phase?

Answer 26

92 kinetochores. There are 46 human chromosomes, each has two sister chromatids with each their own kinetochore.

Question 27

What are possible causes for chromosomes ending up in one daughter cell?

Answer 27

1) Microtubules or their connections with a sister chromatid could break during anaphase. Both sister chromatids would be drawn to one pole and end up in one daughter cell. 2) Micotubules from the same pole could attach to both kinetochores and pull the chromosome to one pole. 3) If cohesins were not degraded, the two sister chromatids would be pulled to the same pole. 4) If a chromosome was never attached to the mitotic spindle, it would be distributed randomly in the new daughter cells. 5) If only one kinetochore becomes attached to a mictotubuli, both sister chromatids would be pulled to that pole.

Question 28

How does the cell ensures correct microtubule attachment to the sister chromatids?

Answer 28

Bipolar attachment is ensured by the tension created when two microtubules pull towards different poles. Without the tension (e.g. when both kinetochores are attached to the same pole) Aurora B kinase phosphorylates Ndc80 and detach microtubules from the kinetochores.

Question 29

What is the difference between mitogens, growth factors, and survival factors?

Answer 29

Mitogens stimulate cell proliferation primarily by triggering G1/S-Cdk activity. Growth factors stimulate cellular growth as in increase in size and production of proteins and organelles and inhibition of degradation of proteins as well as increased uptake of nutrients. Survival factors suppress apoptosis in the cell. Many extracellular signaling factors can function as all three.

Question 30

What are E2F proteins?

Answer 30

E2Fs are transcription factors that can induce expression of factors stimulating entry into S-phase. These include G1/S-cyclins, S-cyclins, and proteins involved in DNA synthesis.

Question 31

How are E2F proteins activated in response to mitogen stimulation of a cell?

Answer 31

Mitogenic stimulation of the cells induces a MAP kinase cascade leading to expression of Myc, which induces expression of the G1-cyclin. Activated G1-Cdk phosphorylates Rb*E2F leading to release of active E2F from Rb. Active G1/S-Cdk and S-Cdk further phosphorylates Rb. Mitogen => MAP kinase cascade => Myc expression => G1-cyclin expression => active G1-Cdk => phosphorylation of Rb(*E2F) => released active E2F => G1/S-cyclin and S-cyclin expression

Question 32

Why is the retinoblastoma protein Rb regarded as a cancer critical protein?

Answer 32

Loss of Rb function alleviates E2F inhibition making it constitutively active. The resulting increase in E2F-stimulted gene transcription cause aberrant cell cycle control and promotes proliferation of cancer cells.

Question 33

Is it true that in order for proliferating cells to maintain a relatively constant size, the length of the cell cycle must match the time it takes for the cell to double in size?

Answer 33

Yes, if the length of the cell cycle is shorter than it takes for the cell to double in size, the cell would get progressively smaller with each division. If the cell cycle is longer, the cells would get bigger and bigger

Question 34

What would happen to a mutant cell that could not degrade M-phase cyclins?

Answer 34

The cell would be unable to divide. They could enter the M-phase but not complete it since removal of M-Cdk activity is necessary for exit from the M-phase

Question 35

What would happen to a mutant cell that always express high levels of p21?

Answer 35

The cells would be unable to divide, they would arrest permanently in G1 because their G1/S-Cdk and S-Cdk complexes would be inactive

Question 36

What do you suppose would happen to a mutant cell that cannot phosphorylate Rb?

Answer 36

The cells would be unable to divide since the E2F proteins would be sequestered in their inactive state bound to Rb. They therefore would not be able to activate transcription of G1/S-cyclin and S-cyclins.

Question 37

How does the cell ensure that every nucleotide in the genome is copied once, and only once during each cell cycle to prevent the damaging effect of gene amplification?

Answer 37

The genome is replicated in the S-phase when the DNA polymerases bind to a replicative origin sites. It can only bind when a prereplicative complex (preRC) has been formed at the origin site. Geminin inhibits the formation of the preRC except in the late M-phase early G1-phase where it is marked for degradation by APC/C. This way the formation of preRC and the binding of DNA polymerase is temporally separated in different phases and can only occur once per cell cycle

Question 38

How is substrate binding and specificity determined for the SCF and the APC/C E3 ubiquitin ligase complexes?

Answer 38

APC/C regulators (Cdc20 and Cdh1) give APC/C specific activity towards different subsets of substrates. F-box proteins are part of SCF complexes, which are responsible for substrate recognition through specific sequence interaction that often involves phosphorylation

Question 39

What is the main difference between mitosis and meiosis?

Answer 39

In meiosis, one round of DNA replication is followed by two rounds of chromosome segregation resulting in four haploid daughter cells. In mitosis, DNA replication is followed by one segregation event resulting in two diploid daughter cells

Question 40

How does genetic diversity in daughter cells arise from meiosis?

Answer 40

After DNA replication, the paternal and maternal homologs each have two sister chromatids and will be in close contact with each other. Homologous recombination events between paired homologs during meiosis I lead to reciprocal exchange of DNA between non-sister chromatids. In meiosis II, the recombinant sister chromatids will be distributed to haploid daughter cells with a random combination of recombinant chromosomes

Question 41

How can activation of p53 arrest a cell in G1?

Answer 41

a) The level of p53 is maintained at a low level in cells by ubiquitin-mediated degradation. When stabilized, p53 accumulates to high levels and stimulates transcriptions of multiple genes, including the gene encoding p21. The p21 protein binds and inactivates the kinase activity of the G1/S-Cdk and S-Cdk complexes, preventing cells from progressing through the G1-S-phase

Question 42

Why is the G1-arrest caused by activation of p53 irreversible in normal cells upon centrosome loss but doesn't happen in cancer cells?

Answer 42

The G1 arrest is irreversible in normal cells because they are caught in a “catch-22 situation”. To cancel the G1 arrest, the cells need to deactivate the loss-of-centrosome induced p53 stabilization by centrosome re-assemble. The cells are, however, unable to proceed to the S-phase where centrosome biogenesis takes place because of the G1 arrest. Cancer cells commonly have loss-of-function mutations in p53. Thus, they are unable to invoke the p53-mediated cell cycle arrest in the first place.

Question 43

Why does some cells undergo apoptosis?

Answer 43

. Incorrect sister chromatid segregation might lead to aneuploidy. In those cases, cellular stress frequently induces apoptosis.

Question 44

What is cohesin?

Answer 44

Cohesin is a protein complex that regulates the separation of sister chromatids during cell division, either mitosis or meiosis. Cohesins hold sister chromatids together after DNA replication until anaphase when removal of cohesin leads to separation of sister chromatids

Question 45

Explain the role of APC/C, Cdc20, separase, cohesin and securin in avoiding segregation errors?

Answer 45

Any kinetochore that is not properly attached to the spindle sends out a diffusible negative signal that blocks the APC/C activation and thus blocks the metaphase-to-anaphase transition. When the kinetochores are properly attached, the activation of the APC/C via the specificity factor cdc20 leads to ubiquitylation and destruction of securin, which normally holds separase in an inactive state. The destruction of securin allows separase to cleave cohesion complexes holding the sister chromatids together. This allows sister chromatid separation and metaphase-to-anaphase transition.

Question 46

Describe an experiment with fluorescent microscopy and antibodies binding the centrosome marker proteins ɣ-tubulin and Cep192

Answer 46

d) Initially, the cells are fixed in ice cold methanol, which preserve the cells and make the plasma membrane permeable for the antibodies. Primary antibodies bind epitopes on target proteins, while secondary antibodies bind the constant domain of the primary antibodies. A fluorophore is covalently attached to the secondary antibodies, which enable visualization and subcellular localization by fluorescence microscopy. The use of antibodies obtained from different species makes it possible to use several fluorophores to localize different thing.