7: Cell Cycle Flashcards
What is aneuploidy?
A condition where the cell or organism has an abnormal number of chromosomes
What are the four phases of the cell cycle (in order)?
M phase (mitosis)
G1 (gap 1)
S phase (DNA synthesis)
G2 (gap 2)
What are PTMs?
Post-translational modifications
Chemical reactions that side-chains undergo that change the nature of the protein
E.g. reactive side chains more likely to undergo PTMs
Increases diversity beyond 20 amino acids
Reversible - can be restored to initial function so makes it variable
What is protein phosphorylation?
A post-translational modification (PTM)
Addition of phosphate group (via ATP) to amino acid side chain with hydroxyl group
Requires specific enzymes which co-ordinate the protein with the ATP and provide the reactive base to initiate the reaction
E.g. serine –> phosphoserine
What are the enzymes required for protein phosphorylation?
Protein kinases
Co-ordinate the protein with ATP and provide the reactive base to initiate reaction
Can be regulated by accessory proteins
What are the two classes of protein kinase used for protein phosphorylation?
Ser/Thr kinases
Tyr kinases (more predominant in eukaryotes than prokaryotes)
What are consensus sequences?
The kinase-specific recognition sequences adjacent to the phospoacceptor residue It is required for protein phosphorylation
Substrate proteins may also bind kinase independent of these sequences to ‘dock’ it
How can protein phosphorylation be reversed?
Via protein phosphatases
What are the two main consequences of protein phosphorylation?
Promotion or impairment of enzyme activity
Promotion or impairment of protein-protein interactions
Dependent on whether the phosphorylation is activating or inhibitory
How does phosphorylation impact human PLK1?
Human PLK1 is a protein kinase
Inactive unless PTM protein phosphorylation occurs
A threonine residue in the activation loop is phosphorylated (via a different protein kinase) which changes the conformation and allows the substrate to bind and therefore be phosphorylated
This is an example of activating phosphorylation (phosphorylation is not always activating, can sometimes be inhibitory)
What key protein regulates mitosis?
Cyclin-dependent kinase (CDK)
What is cyclin-dependent kinase?
Enzyme that regulates mitosis
Active only when bound to a regulatory protein called cyclin
Is both a worker and manager, meaning it regulates other protein kinases like the ‘manager’, but also regulates many proteins directly, like a ‘worker’
What are the four main roles of CDK?
Drives chromosome condensation
Disassembly of nuclear envelope
Mitotic spindle assembly and function
Cytokinesis
What are some examples of substrate proteins of CDK?
Chromosomal proteins
Nuclear lamins
Microtubule-associated proteins
Many others including protein kinases
What is the consensus sequence of substrates of CDK?
Serine, Threonine, Proline, X(Any), Lysine/Arginine (just something basic)
How do cyclin levels change throughout the cell cycle?
They go up and down in waves
Highest CDK activity correlates with when mitosis occurs
What experiment was carried out to prove that cyclin accumulation drives entry into M-phase?
1) Make frog egg cell extract
2) Destroy all mRNA with RNase enzyme
3) Add inhibitor of RNase
4) Add cyclin mRNA and radioactively labelled methionine
5) At different timepoints, analyse new protein synthesis by SDS-PAGE and measure CDK activity
How does cyclin accumulation lead to a positive feedback loop that activates CDK?
CDK activated by activating and inhibitory phosphorylations on different amino acids
Inactive form of CDK is phosphorylated at inhibitory sites by specific kinases
Cdc25 phosphatase counteracts this by removing inhibitory phosphates to activate CDK
When cyclin levels are low, inhibitory phosphorylation predominates and CDK remains inactive
When cyclin levels are high, more CDK is activated by Cdc25
Active CDK then phosphorylates and activates Cdc25, enhancing its activity
Creating a positive feedback loop (dependent on a ‘threshold level’ of cyclin)
What are the four steps within M-phase (mitosis)?
1) Prophase (nuclear envelope still intact), chromosomes condense and become visible
2) Metaphase, chromosomes line up, mitotic spindle forms
3) Anaphase, where sister chromatids are pulled apart
4) Telophase, with cytokinesis, when nuclear envelopes reform and daughter cells are pinched apart
What are isopeptide bonds and why are they different to peptide bonds?
Isopeptide bonds occur between side chain residues of amino acids
Whereas peptide bonds occur between main chain residues
What chemical change causes mitosis to end?
Decreased CDK activity due to decreased cyclin levels
Cyclin protein degraded by proteases through proteolysis via ubiquitylation (PTM)
What is ubiquitin?
A small protein that can bind to proteins via isopeptide bonds
What is ubiquitylation?
The modification of an entire protein when ubiquitin is added via an isopeptide bond
How does ubiquitilation initiate degradation of cyclin and the end of mitosis?
When chromosomes align in metaphase, this activates APC
When APC is activated, it polyubiquitilates cyclin
Ubiquitilated cyclin gets digested by proteasome and is degraded
Destruction of cyclin leads to inactivity of CDK, which therefore prevents mitosis
What is the APC?
Anaphase-promoting complex (APC)
Asssembled by around 19 proteins joined via non-covalent interactions
Causes polyubiquitilation of cyclin which targets it for degradation by proteasomes
How is ubiquitin joined to a target protein?
Ubiquitin has its C-terminus that makes a covalent bond with a lysine residue in the target protein to form an isopeptide bond
Ubiquitin then acts as a flag for further reactions
Polyubiquitilation occurs when multiple ubiquitins are added on top of each other via its own lysine residues
How are sister chromatids kept together during metaphase?
Via cohesin: a protein complex that links the DNA of one sister chromatid to another
For sister chromatids to separate in anaphase, the cohesin rings must break
How are the cohesin rings broken to allow metaphase to move into anaphase?
Separase enzyme is normally inactive because it has an inhibitory bound to it (securin)
When APC is activated at the metaphase-anaphase transition, it ubiquitilates securin
This breaks it via proteolytic degradation (via the same mechanism as ubiquitilation of cyclin) which frees separase to cleave cohesin
How can staining DNA be used for quantitative analysis of DNA content of cells?
1) Stain cells with propidium iodide, a fluorescent dye that binds to DNA. Fluorescence increases when bound to DNA so fluorescence signal is proportional to quantity of DNA
2) Shine a laser on the cells to measure emitted fluorescence
3) Can be used to measure the DNA content of a cell culture and determine what proportion of cells are in each phase of the cell cycle
How does DNA content vary over the cell cycle?
DNA content lowest in G1 - hasn’t duplicated DNA get
Intermediate in S phase - increases gradually as DNA replication occurs
Highest in G2/M phase - DNA is fully replicated
What phase of the cell cycle does DNA replication occur in?
The S phase
What happens in G2 of the cell cycle?
Cyclin levels accumulate
Initiating an abrupt change in CDK activity that initiates the M phase
What is the ORC?
Origin recognition complex
A protein complex that binds to all recognition origins throughout the cell cycle
Inactive for much of the cell cycle because it is phosphorylated
What is the preRC?
Prereplicative complex
A protein complex that forms at repication origins and is essential to initiate DNA replication
Includes ORC and other proteins that load the MCM complex onto DNA
What is the MCM complex?
Protein complex crucial for helicase activity to unwind DNA for replication to occur
This complex only forms once in one cell cycle (during G1) to ensure replication only occurs once
What happens to the preRC during the S phase?
CDK activity is increased, regulated by cyclin proteins
Activated CDKs phosphorylate components of the preRC, which causes:
Activation of MCM to unwind DNA
Prevention of reassembly of preRC during the same cell cycle
Therefore ensuring replication only occurs once
What ensures the preRCs can be formed again in daughter cells after mitosis is finished?
Variable CDK activity
When CDK is active it phosphorylates components of the preRC, preventing them from binding to replication origins, therefore preventing preRC assembly
When activity is low, less phosphorylation occurs, allowing preRC components to bind to the origins and assemble in new daughter cells
What prevents DNA from replicating when it is damaged?
If DNA is damaged, ATR and ATM protein kinases detect it
ATR and ATM activate ‘checkpoint’ protein kinases (Chk1 and Chk2)
Chk1 and Chk2 phosphorylate Cdc25
This inhibits Cdc25
So Cdc25 cannot remove inhibitory phosphates from CDK
And therefore mitosis cannot proceed
Preventing the replication of damaged DNA
When DNA damage has been repaired, Chk1 and Chk2 stop signalling
What prevents anaphase from happening until all sister chromatids are bioriented?
For anaphase to occur, all sister chromatids must be attached to microtubules via their kinetochores to opposite spindle poles in a bipolar fashion
Unattached kinetochores generate a diffusible “wait anaphase” signal
This signal inhibits APC
Inhibition of the APC prevents cyclin degradation and prevents cohesin cleavage
When there are no unattached kinetochores, the signal is destroyed
And anaphase can proceed again
What are the three main reasons why cell division is necessary?
1) Development of the organism, including differentiation into different cell/tissue types
2) Renewing cells/tissues that turn over rapidly (e.g. intestine, blood)
3) Repairing damaged tissue after injury or infection
What prevents cell proliferation?
The late G1 checkpoint
Called ‘start’, ‘restriction point’, or ‘commitment point’
Once cells get past this point, it is commited to division
Later checkpoints are only about error correction
What is G0?
The non-dividing, resting state that some cells enter from G1
These cells are active but not preparing for division
How do cells progress through the start checkpoint during late G1 phase?
Mitogens bind to cell surface receptors early in G1, triggering intracellular signalling that increases G1/S cyclin expression
This triggers a pathway that unleashes activity of a transcription factor that promotes S phase and drives DNA synthesis
What are mitogens?
Outside signals that bind to a receptor to promote cell proliferation via complex pathways
What is contact inhibition?
When cells grow, e.g. in wound healing, they form a monolayer
Once the cells form a monolayer, they make attachments to each other
Which seems to release a signal that prevents further growth
Many cancer cells seem to lose the ability to send or receive this signal
What are telomeres?
Telomeres are repetitive DNA sequences at the ends of linear chromosomes that protect the chromosome ends but shorten slightly with each round of DNA
How do telomeres act as a barrier to unlimited cell proliferation?
Telomeres are repetitive DNA sequences at the ends of linear chromosomes that protect the chromosome ends but shorten slightly with each round of DNA replication because the replication machinery can’t fully copy telomeres
After 25-50 replications, telomeres become critically short
The cells sense this a DNA damage and trigger programmed cell death
What is telomerase?
An enzyme that can extend telomeres
It isn’t active in most somatic cells, but is in germ cells, stem cells, and many cancer cells
How does differentiation limit cell proliferation?
Stem cells divide to produce one identical stem cell (self-renewal), and one differentiating cell with limited developmental potential
As cells differentiate, they lose the ability to divide indefinitely because specific genes controlling proliferation are no longer expressed
This means the stem cell pool stays constant, and total proliferation capacity is limited
This prevents uncontrolled expansion and helps to maintain proper tissue size and function
What problems does cancer cause with the cell cycle?
Inappropriate expression of positive signals when no signal was received
Failure to produce/respond to negative signals
Failure to senesce (die)
How do multiple mutations contribute to cancer?
Mutations can synergise at multiple points in growth and division pathways, greatly increasing risk of cancer
How can mitogens contribute to cancer?
Excess mitogen signalling or mutations in mitogen receptors can driver hyper-proliferation, potentially contributing to cancer
What is the role of Ras in cancer?
Activating mutations in Ras are found in about 25% of all human cancers, promoting constant growth signalling
What is the role of Rb in cancer?
Rb mutations disrupt control of the cell cycle and are found in 15-30% of many cancers, and 90-100% of retinoblastoma and small-cell lung cancers
What is p53 and why is it important?
P53 is a transcription factor known as the “guardian of the genome” It is mutated in over 50% of all human cancers
How is p53 normally regulated?
In healthy cells, p53 is unstable and quickly destroyed via ubiquitation, keeping its levels low
What activates p53 and what does it do?
Cell stress inhibits p53 degradation, causing its activation
Cell stress can include DNA damage, low oxygen, telomere shortening, etc.
Mild damage causes p53 to pause the cell cycle for DNA repair
Severe damage leads to p53 triggering apoptosis
What happens when p53 is lost?
Without p53, cells can’t sense or respond to DNA damage, leading to unprepared DNA, chromosomal abnormalities, and increased cancer risk
