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SMP - Genetics > Lecture 13 - The Cell Cycle > Flashcards

Lecture 13 - The Cell Cycle Flashcards

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1
Q

How long does the S phase of the cell cycle take?

A

10-12 hours

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2
Q

How long does the M phase of the cell cycle take?

A

Less than 1 hour

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3
Q

What 2 events does the M phase of the cell cycle include? Describe each.

A
  1. Mitosis = nuclear division

2. Cytokinesis = cytoplasmic division

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4
Q

What are the 4 phases of the cell cycle?

A
  1. G1
  2. S
  3. G2
  4. M
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5
Q

During which phase of the cell cycle does DNA replication occur?

A

S

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6
Q

How long does the G1 phase of the cell cycle take?

A

6-12 hours

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7
Q

How long does the G2 phase of the cell cycle take?

A

3-4 hours

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8
Q

What happens during G1 phase?

A

RNA and protein synthesis

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9
Q

What happens during the G2 phase?

A

RNA and protein synthesis

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10
Q

What happens during the S phase?

A

DNA replication + RNA/protein synthesis

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11
Q

Describe the cells in the G0 phase.

A

Terminally differentiated cells withdraw from the cell cycle indefinitely in G0

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12
Q

What does the length of all of the cell cycle stages depend on?

A

Age of the organism and other signals

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13
Q

What is the restriction point?

A

Check-point of the G1 phase past which it is committed to the S phase

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14
Q

How do a lot of researchers try to affect what phase a cell is in?

A

Getting G0 phase cells to re-enter the cell cycle to replace damaged or dead cells

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15
Q

What is an example of plasticity of dividing cells?

A

During the growth of the egg in the ovary, cells are experiencing G phases without dividing and mimicking G2 and then upon fertilization then divide without any G phases (so the cells get progressively smaller with each division, and the embryo remains the same size) to reach a critical mass with a critical number of cells to start differentiation

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16
Q

How can we study the cell cycle? How does this technique work?

A

Flow cytometer allows separation/purification of cells depending on what phase they are in (eg: amount of DNA they have)

Flow cytometer is used to sort cells according to their fluorescence and cells are stained with a dye that becomes fluorescent when it binds to DNA, so that the amount of fluorescence is directly proportional to the amount of DNA in each cell. The cells fall into three categories:

  1. Those that have an unreplicated complement of DNA = G1
  2. Those that have a fully replicated complement of DNA = G2 or M phase
  3. Those that have an intermediate amount of DNA = S phase
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17
Q

How did researchers study the cell cycle prior to the invention of the flow cytometer?

A

They would arrest cells in certain phases to identify certain proteins that were expressed during each phase

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18
Q

How can we birthdate cells? What labels are used?

A

Use pulses of radiolabelling nucleotides during a certain period then you can later assess when certain cells were born

Labels:

  1. 3H-thymidine
  2. BrdU-labeling (uridine)
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19
Q

What is the purpose of birthdating cells?

A

Figuring out their half life

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20
Q

How were we able to do restrospective birthdating of human neurons?

A

In the 60s we were doing a lot of nuclear testing so the 14C levels in humans were much higher so the levels are directly proportional to the birth of the neurons (aka the age of a person)

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21
Q

What does the amount of 14C in neurons of the human cerebral cortex correspond to? What does this show?

A

The amount of 14C found in the atmosphere at the time of birth of the individual. This shows that there is minimal turnover of neurons during postnatal and adult life.

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22
Q

What are the 2 models that explain how the cell cycle is regulated? Which one is now widely accepted?

A
  1. Dominos model

2. Washing machine model***

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23
Q

Explain the dominos model of cell regulation.

A

Events in the cell cycle would follow each other: when one phase completes, another begins

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24
Q

Explain the washing machine model of cell regulation.

A

Central controller that coordinates the events of the cell cycle

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25
Q

What is the central controller of the cell cycle? What does it ensure?

A

A protein kinase: Cyclin-dependent protein kinase (Cdk)

Ensures certain criteria are met before certain phases are entered

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26
Q

How do cell cycle checkpoints operate? Explain what this means.

A

Through negative control: the default position is to arrest/not proceed unless certain criteria are met

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27
Q

What does the start checkpoint check? Where is it?

A

Is the environment favorable?

G1/S

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28
Q

What does the G2/M checkpoint check?

A
  1. Is all DNA replicated?

2. Is the environment favorable?

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29
Q

What does the metaphase/anaphase transition check?

A

Are all chromosomes attached to the spindle?

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30
Q

What are the 3 checkpoints of the cell cycle?

A
  1. Start checkpoint
  2. G2/M checkpoint
  3. Metaphase/anaphase transition
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31
Q

What is the major checkpoint in yeast?

A

Start checkpoint

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32
Q

What is another name for the start checkpoint?

A

G1 checkpoint

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33
Q

What is the major checkpoint in eukaryotes?

A

G1 checkpoint

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34
Q

What kind of kinase is Cdk?

A

Ser/Thr protein kinase

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35
Q

What are the 2 roles of the cyclins?

A
  1. Activate Cdks or put them in a position where they are ready to be activated
  2. Direct the Cdks towards specific substrates
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36
Q

Are there multiple types of Cdks in mammals?

A

YUP

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37
Q

What are the 4 classes of cyclins?

A
  1. G1/S cyclins
  2. S-cyclins
  3. M-cyclins
  4. G1-cyclins
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38
Q

What is the role of G1/S-cyclins? When do they bind Cdks?

A

Activate Cdks in late G1 to help trigger progression through the start checkpoint to commit to cell cycle entry

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39
Q

During what cell cycle phase G1-S-cyclins levels fall? When exactly?

A

S phase, right at the start

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40
Q

What is the role of S-cyclins? When do they bind Cdks?

A

Bind Cdks soon after progression through the start checkpoint and help stimulate chromosome duplication and some early mitotic events

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41
Q

During what cell cycle phase S-cyclins levels fall? When exactly

A

M: metaphase/anaphase transition

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42
Q

What is the role of M-cyclins?

A

Activate Cdks to stimulate entry into mitosis at the G2/M checkpoint

43
Q

During what cell cycle phase M-cyclins levels fall? When exactly?

A

Mid-M phase (shortly after metaphase/anaphase transition)

44
Q

What is the role of G1-cyclins?

A

Help govern activities of the G1/S cyclins

45
Q

Do most cells use all 4 classes of cyclins?

A

NOPE, usually they use all except G1-cyclins

46
Q

Do the Cdk concentrations fluctuate through the cell cycle?

A

NOPE

47
Q

How does [Cdk] compare to [cyclins]?

A

[Cdk] exceed the amounts of cyclins

48
Q

When do M-cyclin-Cdk complexes form?

A

During G2 but are held in an inactive state until the end of the G2 phase

49
Q

What protein regulates the metaphase/anaphase transition?

A

The APC/C regulatory protein

50
Q

Are cyclins sufficient for Cdk activation?

A

NOPE

51
Q

Other than cyclins, what else do Cdks need for activation?

A

Other kinases and phosphatases

52
Q

Describe the full activation of Cdks. 5 steps

A
  1. In the absence of cyclin binding, there is a T loop of the Cdk that occupies the active site (internal inhibitory peptide sequence)
  2. Cyclin binds changing the confirmation of the Cdk removing the T loop from the catalytic domain = Cdk partly active
  3. Wee1 inhibitory kinase phosphorylates Cdk blocking the ATP binding site = inactive Cdk
  4. Activating phosphorylation on T loop by Cdk-activating kinase (CAK) = inactive Cdk
  5. Active cdc25 dephosphorylates Cdk by removing inhibitory P group = fully active Cdk
53
Q

How does Cdk T loop phosphorylation by CAK fully activate the Cdk?

A

Changes T loop shape, enhancing Cdk’s ability to bind substrates

54
Q

What kind of kinase is CAK?

A

Threonine kinase

55
Q

For what do we use and create mutations?

A

Use and creation of mutations is critical to identify roles of proteins

56
Q

What are 2 cell division mutants?

A
  1. cdc mutants = cell division cycle mutants

2. wee mutants

57
Q

How do cdc mutants act?

A

They arrest at a specific point in the cell cycle because they lack a gene product required to get past a checkpoint

58
Q

How do wee mutants act?

A

They divide at a smaller size than normal (and keep getting smaller with each cell division) because they lack a gene product that inhibits passage through a size checkpoint

59
Q

What is the effect of Wee1 kinase on Cdk?

A

Inhibitory phosphorylation usually prior to mitosis

60
Q

What is the effect of Cdc25 phosphatase on Cdk?

A

Activating dephosphorylation (removing inhibitory phosphate group) usually prior to mitosis

61
Q

What is the role of the wee1 kinase and cdc25 phosphatase on Cdk? What would happen if there was a mutation in wee1 kinase? What would happen if there was a mutation in cdc25 phosphatase?

A

The wee1 kinase blocks activity and when the environment is ready the cdc25 phosphatase activates it to trigger mitosis

  • Mutation in wee1 kinase: the cell will keep dividing
  • Mutation in cdc25 phosphatase: cell cycle arrest
62
Q

How are ALL cyclins degraded? What regulates this

A

Cyclical proteolysis governed by targeted ubiquination

63
Q

What does APC/C stand for? 2 names

A

Anaphase-Promoting Complex = Cyclosome

64
Q

What kind of enzyme is APC/C?

A

Ubiquitin ligase

65
Q

Describe the control of cyclical proteolysis of M and S cyclins by APC/C. 3 steps

A
  1. Inactivated APC/C is activated by Cdc subunit which recognizes specific sequences on cyclins
  2. With the help of two additional proteins called E1 and E2, the active APC/C transfers multiple ubiquitin molecules onto the Cdk-M or S-cyclin complex
  3. The polyubiquitylated Cdk-M or S-cyclin is then recognized and degraded in a proteasome
66
Q

What cdc subunit will activate APC/C to degrade Cdk-M-cyclin complex?

A

Cdc20

67
Q

Why would the cell cycle arrest if the cdc subunit activating the APC/C was mutated?

A

In addition to degrading the M-cyclins, the APC/C degrades securin, which protects the proteins that hold sister chromatids together => destruction of securin activates a protease that separates the sister chromatids and unleashes anaphase

IF YOU DO NOT DEGRADE SECURIN, THE SISTER CHROMATIDS WILL STAY ATTACHED = CELL CYCLE ARREST

68
Q

When does Cdk dephosphorylation (aka deactivation) occur during the cell cycle?

A

After the metaphase-anaphase transition

69
Q

How do cell cycle proteins work together?

A

They form a robust network which operates essentially autonomously to activate a series of biochemical switches, each of which triggers a specific cell cycle event

70
Q

What proteins does APC/C primarily ubiquitylate?

A

Proteins involved in the exit from mitosis: S and M cyclins and securin

71
Q

What are the 2 roles of SCF? What kind of enzyme is it?

A

Ubiquitin ligase which catalyzes

  1. Ubiquitynation of regulatory proteins involved in G1 control including some Cdk Inhibitor Proteins (CKIs)
  2. Ubiquitynation of p57 leading to the activation of G1-S-Cdk complexes
72
Q

How will DNA damage affect the cell cycle?

A
  1. Inhibit G1/S-Cdks complexes
  2. Inhibit S-Cdks complexes
  3. Inhibit M-Cdks complexes

(Depending on phase in which DNA damage is found)

73
Q

How will unreplicated DNA affect the cycle?

A

Inhibit M-Cdks complexes

74
Q

How will a chromosome unattached to the mitotic spindle affect the cell cycle?

A

Inhibit APC/C

75
Q

What would inhibit DNA re-replication?

A

M-Cdk complexes

76
Q

Are all cell cycle inhibitory mechanisms present in all cell type? Provide an example.

A

NOPE

Eg: many are missing in early embryonic cell cycles

77
Q

How does S-Cdk affect the cell cycle exactly?

A

Stimulates the formation of replication forks and the initiation complex

78
Q

What cell cycle regulatory protein levels are necessary for the pre-replicative complex to form?

A
  • Low Cdk

- High APC/C

79
Q

How do the cell cycle regulatory proteins ensure that replication only happens once during a cell cycle?

A

Increase in S-Cdks => Formation of replication forks in S phase => S-Cdks trigger destruction of cdc6 and inactivates ORC (both by phosphorylation) => disassembly of the prereplicative complex, which does not reform at the origin until M-Cdks are inactivated and APC/C is activated at the end of mitosis, aka until the following G1

80
Q

What are the 3 components of the pre-replicative complex?

A
  1. Cdc6
  2. Cdt1
  3. Mcm
81
Q

What is mcm?

A

DNA helicase

82
Q

How/When is Cdt1 inactivated?

A

By the protein geminin during the S phase

83
Q

What are the 3 feedback loops of the full activation of a Cdk?

A
  1. Positive feedback of fully active Cdk on Wee1 inhibitory kinase to inhibit it by phosphorylating it
  2. Positive feedback of fully active Cdk on inactive Cdc25 to phosphorylate and active it
  3. Negative feedback loop of fully active Cdk triggering DBRP activation to trigger ubiquination of cyclin
84
Q

When is Cdc25 active?

A

When it’s phosphorylated

85
Q

When is Wee1 kinase active?

A

When it’s NOT phosphorylated

86
Q

What does DBRP stand for?

A

Destruction Box Recognizing Protein

87
Q

What cascade leads to the synthesis of cyclins and Cdks to trigger the passage from G1 to S? What does this cascade also synthesize?

A

Growth factors, cytokines, and mitogens trigger a MAPK cascade leading to the phosphorylation of the nuclear transcription factors Jun and Fos

Also synthesizes E2F transcription factor for promoting the expression DNA replicative enzymes

88
Q

What are 2 gene types that regulate the cell cycle? Describe each.

A
  1. Proliferation genes = proto-oncogene

2. Antiproliferation genes = tumor suppressor

89
Q

What are the 3 functions of proliferation genes?

A
  1. Promote cell growth
  2. Promote assembly of the cell cycle control system
  3. Drive the cell past the G1 checkpoint
90
Q

What is the function of antiproliferation genes?

A

Halt the cell cycle control system

91
Q

What is another name for antiproliferation genes?

A

Tumor suppressor genes

92
Q

What are oncogenes?

A

Overexpressed or mutation activated proliferation genes causing cancer

93
Q

What is an example of a antiproliferation gene? How does its product work? What happens when it’s mutated?

A

Retinoblastoma (Rb) coding gene

Rb binds to the transcription initiation factor EF2, inhibiting it

It is phosphorylated to be inactivated, thereby releasing/activating the initiation factor, which is now available to trigger transcription of genes involved in S phase, like G1/S-cyclins and S-cyclins, promoting DNA synthesis

When mutated, the transcription initiation is always active even when it shouldn’t be = uncontrolled cell growth

94
Q

What is a mitogen?

A

Factor that stimulates cell division

95
Q

What is Myc?

A

Immediate early gene product of a mitogen activated MAPK cascade that acts as a transcription factor and is involved in growth and mitosis

96
Q

What is E2F?

A

Transcription initiation factor

97
Q

What is Myc’s overall role? What 3 activations does it trigger to achieve this.

A

Overall role is to activate E2F to trigger cell entry into S phase

  1. Activates cyclin D gene, a G1-cyclin => Rb phosphorylation
  2. Activates SCF subunit gene => increasing p27 degradation => G1-Cdk activation => Rb phosphorylation
  3. Activates E2F gene
98
Q

Effect of Rb phosphorylation?

A

Increased E2F activity

99
Q

What cascade promotes cell growth in G phases?

A

Growth factor activated PI 3-K cascade leading to the activation of eIF4E and S6 kinase, both increasing mRNA translation

100
Q

What is nutritional cell-cycle control? What organisms use this?

A

Cells depend on growth factors, mitogens, cytokines to tell the cell that there are sufficient nutrients and the cell has the proper size before dividing, so the G phases have a certain plasticity to last longer if needed

Advanced eukaryotes

101
Q

What happens without nutritional cell-cycle control? Why?

A

Cells depend on a certain amount of time passing before dividing so the mass of the cells decreases over time because the G phases are not as effective because not enough nutrients

102
Q

How is geminin (which inactivates Cdt1: part of the pre-replicative complex) degraded?

A

APC/C

103
Q

What are the 3 positive feedbacks in the pathway of the retinoblastoma (Rb) coding gene?

A
  1. E2F positive feedback on itself
  2. G1-S-Cdk complexes phosphorylate Rb
  3. S-Cdk complexes phosphorylate Rb
104
Q

How does APC/C recognize which cyclins to ubiquitinate?

A

The cdc that binds to it directs it

SMP - Genetics (16 decks)

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