The Cell Cycle

Question 1

Give an overview of the eukaryotic cell cycle?

Answer 1

Chromosomes are pulled to each pole of the mitotic spindles before being reformed into daughter cells
All contents need to be replicated - cell organelles
All cells arise by division of parental cells
Cell viability and integrity depends on accurate duplication and segregation
The genome must be correctly copied (once) and equally divided between daughter cells
There must be protection against replication of damaged DNA

Options for a cell - proliferation, differentiation, rest or die

Question 2

What are the stages of the cell cycle?

Answer 2

G1 - cell growth and expansion (9 hours)
S - DNA replication (10 hours)
G2 - Preparation for mitosis (4-5 hours)
Mitosis

Question 3

What are the stages of mitosis?

Answer 3

Prophase - chromosome condensation, spindle formation, nuclear envelope breakdown
Metaphase - chromosome alignment
Anaphase - chromosome separation
Telophase - nuclear envelope formation, chromosome de-condensation
Cytokinesis - contractile ring separates into daughter cells

Question 4

What are some methods of studying the mammalian cell cycle?

Answer 4

Detection of S-Phase cells - can use ‘pulse-labelling’ with nucleotide analogue BrdU

Cell synchronisation using reversible inhibitors
Colcemid binds microtubules – inhibits M phase
Thymidine or hydroxyurea - inhibit DNA replication in S phase

DNA synthesis using bromodeoxyuridine (BUdR, nucleotide analogue) and anti-BUdR antibodies

DNA content using flow cytometry (FACS) - a laser that aligns single cells in the beam
They are permeabilised and stained (e.g. SYBR green)
The laser can then measure the fluorescence in the cells
This technique can also be used to see the effect of drugs on the cell cycle e.g. Retinoids (fenret)

Question 5

What systems can be used to study the cell cycle?

Answer 5

Cell fusion between cells in different stages of the cycle - uses polyethylene glycol to fuse plasma membranes (Leland Hartwell)
Frog and amphibian eggs and embryos - led to discovery of maturation or mitosis promoting factor or MPF (Tim Hunt)
Fission and budding yeast - led to discovery of cell division control or cdc genes (Paul Nurse)

Question 6

Describe Hartwell and Nurse’s experiment - the effect of mutation of the CDC gene?

Answer 6

At a permissive (low) temperature - the cells grew normally
Restrictive (high) temperature - the cells were blocked in a particular phase of the gene, but they continue to grow physically in G1 phase
Therefore CDC - cell division cycle genes are code for proteins which regulate cell cycle events

Question 7

Describe Nurse’s experiment - cloning the defective CDC gene in yeast?

Answer 7

He transformed the yeast within a mouse DNA library
The sequence of from mice alignment at 62% - showed eukaryotic cell cycle control mechanisms are highly conserved from yeast to humans (found through sequence alignment)

Question 8

Describe Hunt’s experiment - purifying MPF protein?

Answer 8

MPF protein (maturation promoting factor) is a heterodimer that stimulates cell division
Contains - 1 molecule of cyclin B + 1 molecule of cdc2
Cdc2 is a cyclin dependent kinase (CDK)
Cdc2 = cdk1

Question 9

What proteins are involved in the regulation of the G2/M transition in mammalian cells?

Answer 9

Cdc2 - a protein kinase (cell division cycle gene 2)
CyclinB - a subunit associated with cdc2 kinase protein that ‘cycles’

Regulatory proteins
Cdc25C - a protein phosphatase (cell division cycle gene 25)
Wee1 - a protein kinase (makes cells small)
CAK - a protein kinase (Cdk activating kinase)

Question 10

What are the changes in specific protein levels within the G2/M transition?

Answer 10

Cdc2 protein - remains stable and doesn’t vary
CyclinB - increased up until G2/M transition and then rapidly disappears
MPF activity - shoots up at G2/M phase and rapidly declines

Question 11

Describe the sites of Cdc2 phosphorylation?

Answer 11

Wee1 and CAK phosphorylate cdc2
Both depend on cyclin bound cdc2

Wee1 phosphorylation occludes the ATP binding site - inactivating
CAK phosphorylates at the substrate binding site - activating

Question 12

Describe Cdc2 phosphorylation within the cell cycle of yeast?

Answer 12

Cdc2 is differentially phosphorylated during the cell cycle
M-phase
The active form of cdc2 is bound to CyclinB - it is unphosphorylated at Y15 and phosphorylated at T161 = cdc2 kinase activity
This is mediated by CAK - serine/threonine protein kinase

G1-phase and S-phase
T161 is dephosphorylated and CyclinB is degraded = inactive form

G2-phase
Both Y15 and T161 are phosphorylated - using Wee1 and CAK - CyclinB is also bound to allow the phosphorylation = inactive
Cdc25c (protein phosphatase) - this dephosphorylates the Y15 = active cdc2

Question 13

What are the changes in enzyme activity during the cell cycle of yeast?

Answer 13

CAK activity - remains the same throughout the cycle
Cdc25 activity - peaks just before M
Wee1 - has an inverse relationship with cdc25 activity - as this inhibits cdc25

Question 14

What does cdc2 activation in yeast allow?

Answer 14

The mechanism allows rapid switching

It is brought about by positive and negative feedback

Question 15

What are the Cdk/cyclins involved in mutlicellular/higher eukaryotes?

Answer 15

There are many cyclin dependent kinases - operating at different phases of the cell cycle
G2/M transition - Cdk1/cyclinB
G1-phase - Cdk4/cyclinD
G1/S transition - Cdk2/cyclinE
S-phase - Cdk2/cyclinA

Cdc2 (yeast) = Cdk1 (mammalian

Question 16

What mediates the G2/M phase transition in higher eukaryotes?

Answer 16

Anaphase promoting complex (APC)
Contains a ubiquitin ligase that targets CyclinB for proteolysis
It also degrades the protein (securin) that links sister chromatids

Question 17

What controls the G1/S transition?

Answer 17

Cdk2/cyclinE activity is controlled in a similar way as cdk1/cyclinB
Cdk2 protein - remains constant
Cyclin E protein - peaks at G1/S transition

Question 18

Describe the G1-S checkpoint?

Answer 18

Here there is a transcriptional regulatory mechanism - mediated by cyclins and the inhibitory protein Rb
Rb is a tumour suppressor, mutated/deleted in retinoblastoma

E2F is a transcription factor that activates transcription of S phase genes (coding for proteins involved in DNA synthesis), cyclin E and its own expression
It is negatively regulated by hypo-phosphorylated Rb (recruits histone deacetylase, HDAC; leads to transcriptional repression)

Question 19

What is another control mechanism in mammalian cells?

Answer 19

Cyclin-dependent kinase inhibitors (CDKI)
Two major families
CIP (CDK inhibitory proteins) e.g. p21, p27 and p57
INK (Inhibitor of cdk4) e.g. p16

May act as S phase inhibitors, degraded by proteolysis in late G1
P21 transcription activated by p53 in the DNA damage response – arrests cycle in G1

Question 20

What are the checkpoints in the mammalian cell cycle?

Answer 20

G1 - DNA damage
G2 - Unreplicated or damaged DNA
M - Chromosome misalignment

Question 21

Describe DNA damage response?

Answer 21

Protein kinase ATM phosphorylates p53
P53 is a transcription factor (53kDa) - frequently lost/mutated in multiple cancers
P53 is normally rapidly turned over - with levels increasing in irradiated cells

Phosphorylation blocks binding by MDM2, a ubiquitin ligase that targets p53 for proteolysis
P53 activates p21 transcription, inhibits cdk-cyclin complexes, arrests cells in late G1 - allows damage to be repaired

Very high levels of p53 activate apoptosis – programmed cell death
ATM also activates Chk2, a protein kinase involved in mitotic checkpoint control, inhibiting entry to M phase

Question 22

What is a level of control unique to mammalian cells?

Answer 22

Growth factors
Stimulation of growth-arrested cells (Go) with serum (or growth factors) leads to rapid entry into S phase (DNA replication)
Examples - epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)
They stimulate signal transduction pathways - G-proteins or TKR signalling
Lead to transcription of “early response genes”
E.g. c-jun, c-fos, c-myc (transcription factors)

Their expression can depend on serum/other inhibitors of delayed response genes
Mutation or over-expression of early response genes found in certain cancers (leads to stimulation of cell cycle) - they act as oncogenes
Transcription factor activity of early response gene products leads to activation of “delayed response genes”