The Cell Cycle Flashcards
What is the different types of division?
*Cloning cells of a given type to make tissues
*Making cells of different types (differentiation) – might involve asymmetric divisions
*Making cells with half normal DNA content (meiosis)
What is the purpose of the cell cycle?
To allow a cell to reproduce
Why is the cell cycle important?
*Required for growth, development, and procreation
*High fidelity required to ensure stable inheritance of cell and organism characteristics
*Most be controlled to allow development and prevent disease
What needs to happen to carry out a cell cycle?
Chromosomes need to be duplicated
Other organelles need to copied
Cells need to grow
Chromosomes need to be segregated accurately
Cell needs to physically divide
What are the basic stages of the cell cycle?
G1: Gap 1
S: Synthesis
G2: Gap 2
M: Mitosis
G0: resting state
What is G1 ?
In the cell cycle
- Deciding if conditions are right for a full cell cycle
- Growing and preparing for DNA synthesis
What is the S phase?
Replicating DNA and centrosomes
What is the G2 phase?
Deciding if conditions are right for mitosis
What is the M phase?
Chromosome segregation and cytokinesis.
What is the G0 phase?
Cells not in the cell cycle
- Terminally differentiated cells
- Quiescent cells
- Senescent cells
What are Quiescent cells?
Quiescent cells are in an inactive stage.
Cells enter into the quiescent state due to lack of nutrition and growth factors.
What are the characteristic of Quiescent cells?
Quiescent cells are characterized by a low RNA content, lack of cell proliferation markers and increased label retention, indicating low cell turnover.
What are Senescent cells?
A senescent cell is one whose life cycle has come to a permanent end.
What drives the cell cycle?
Cyclin-dependent kinases (Cdks).
What are Cyclin-dependent kinases (Cdks)?
- Protein kinases that transfer a phosphate onto their substrates
- Act as “master regulators”
- Have multiple target proteins to control numerous processes in the cell cycle
How are Cyclin-dependent kinases (Cdks) activated?
Cdks have little activity by themselves, but they are activated by Cyclin proteins.
How do Cyclin-dependent kinases (Cdks) drive the cell cycle?
Cyclins influence the substrate specificity of Cdks.
When are G1/S-cyclin levels at their highest?
G1 phase.
When are S-cyclin levels at their highest?
G1 to M phase.
When are M-cyclin levels at their highest?
G2 to M phase.
What are some Additional Cdk regulators?
Upstream kinases
Phosphate
Cdk inhibitory proteins (CKIs)
What did Yoshio Masui discover in 1971?
Identified a cytoplasmic factor (MPF) that could induce cell division in frog oocytes
What did Leland Hartwell discover in 1974?
Conducted screens in budding yeast that identified Cell Division Cycle (cdc) mutants including Cdk1 (Cdc28)
What did Sir Paul Nurse discover in 1987?
Identified and characterised Cdk1 (Cdc2) in fission yeast, and cloned human Cdk1 by complementation
How can we study Cell Division Cycle (cdc) mutant cells?
Temperature sensitive (ts) mutants
Track cell cycle by size and budding
What are Temperature sensitive (ts) mutants?
Mutations that allow gene products to function at low temperature, but not higher temperature.
What makes Cyclin levels oscillate?
Mechanisms controlling synthesis include changes in transcription and translation rate, which vary depending on cell type.
What are the APC/C signals?
The APC/C signals degradation of M-Cyclin to end mitosis and initiate cell division.
How do APC/C signals lead to degradation of M-Cyclin?
*The APC/C is a ubiquitin ligase
*It covalently attaches the small protein Ubiquitin to client proteins such as M-Cyclin
*Ubiquitinylation is a tag for protein degradation
What is the function of SCF signals?
SCF signals degradation of CKIs to promote G1-S transition.
How do SCF signals lead to the degradation of CKIs?
*SCF is a ubiquitin ligase
*It covalently attaches the small protein Ubiquitin to client proteins such as CKIs (Cdk inhibitor proteins)
How is cell cycle fidelity maintained?
Cyclin oscillations provide timing for the successive phases of the cell cycle.
Checkpoints.
What are cell cycle checkpoints?
Checkpoints are monitoring systems that check if conditions are right before allowing the next phase to occur.
What does mitogen do within the cell cycle?
Promotes G1/S-cyclin synthesis.
What happens within the cell cycle when DNA is damaged?
Inhibits cyclin activity by phosphoregulation or CKI.
What happen within the cell cycle when chromosomes become unattached?
Prevents M-cyclin destruction.
What is the G1 checkpoint (also known as restriction point or the START)?
*Nutritional conditions suitable
*Proliferation signals
*DNA damage has been repaired
What is the G2 checkpoint?
*DNA damage been repaired
*DNA replication complete
*Cell big enough
What is the DNA damage checkpoint?
*Budding yeast cells in G2 normally arrest if their DNA is damaged with X-rays.
*Rad9 mutant yeast do not delay in G2 after. *DNA damage and they continue to proliferate with damaged DNA and eventually die.
Are Rad9 mutant yeast defective in DNA damage repair?
Rad9 is part of a checkpoint response, not part of the DNA repair response.
What is the Mitotic or Spindle Assembly Checkpoint?
Are chromosomes (properly) attached to
the spindle?
What happens once the Mitotic or Spindle Assembly Checkpoint is satisfied?
*The APC/C is activated to degrade Cyclin B
*The cells exit metaphase into anaphase
What happens if a checkpoint cannot be satisfied?
*Cells will resume the cell cycle if errors or damage can be fixed.
-OR-
*Things cannot be corrected in a timely way.
What kinds of things cannot be corrected in a timely way, for the cell to get through the cell cycle checkpoint?
Cells can withdraw from the cell cycle (senescence)
- Terminal exit from cell cycle
- Allows cell to remain part of tissue but it will not proliferate
Or:
Cells can undergo programmed cell death (apoptosis)
- Removes cell from organism
What is the CDK that is involved in the G1 phase?
CDK4 & 6
What is the CDK that is involved in the G1/S phase?
CDK2
What is the CDK that is involved in the S phase?
CDK2, & CDK1 (CDC2)
What is the CDK that is involved in the M phase?
CDK1 (CDC2)
What is the cyclin involved in the G1 phase?
Cyclin D
What is the cyclin involved in the G1/S phase?
Cyclin E
What is the cyclin involved in the S phase?
Cyclin A
What is the cyclin involved in the G2/M phase?
Cyclin B
What are the main checkpoints in the cell cycle?
- Restriction point (or START in yeast)
- G2/M (DNA damage) checkpoint
- Mitotic checkpoint
How do cells control their CDK-cyclin kinase activity?
- Transcription
- Cyclin-dependent kinase inhibitors (CKIs) and others
- The antagonized phosphorylation and
dephosphorylation - Ubiquitin-mediated proteolysis
What is Myc?
A transcriptional factor, and one of the early G1 genes.
What does Myc do within the G1-S transition?
*Can react to the mitogens, activates and increases the transcription of several genes, including: cyclin D
*SCF ubiquitin ligase (for protein proteolysis)
What are the stages of G1 - S transition?
- The early G1 genes determine the G1/S transition
- CKs-dependent G1/S checkpoint activation
- CDC25-mediated inhibitions by TGFb & DNA damage pathways
- SCF E3 ligase-mediated inhibition
What is at the 5’ end of a DNA molecule?
Phosphate
What is at the 3’ end of a DNA molecule?
hydroxyl group
What direction is DNA read in?
5’ to 3’
What bonds are in the sugar phosphate backbone?
Phosphodiester bonds
How many hydrogen bonds are between adenine and thymine?
2
How many hydrogen bonds are between guanine and cytosine?
3
Which of the bases are purines?
Adenine and guanine
Which of the bases are pyrimidine?
Thymine and cytosine.
What are the key biochemical activities of DNA replication?
Initiation
Elongation
Termination
The end-replication
problem
Outline the process of recognition and activation of initiation.
- DNA replication begins at the origin (oriC) of replication.
- The oriC will be recognized by ORC (Origin recognition complex) for DNA unwinding.
- To preserve the genome’s integrity, each replication origin can only be activated once per cell cycle.
Outline the process of licensing and assembly of the replication complex of initiation.
- Licenses form an origin for a single initiation.
- Geminin binds to Cdt1
- This prevents it from loading Mcm complex onto the origin DNA.
- Geminin is degraded by APC/C mediated ubiquitin proteolysis.
- This releases Cdt1.
- This enables ORC-CDC6-Cdt1 complex recruit Mcm.
- Replication starts when CDK2-Cyclin A (CycA) phosphorylates the MCM2-7 hexamer.
- This form the replicative CMG helicase with the GINS complex and CDC45.
- The re-accumulation of Geminin in the S phase inhibits the assembly of new prereplication complexes until after the next mitosis.
What are licenses?
Prereplication complexes, origins for a single initiation events.
What is geminin?
Origin licensing regulator.
What is the function of geminin?
Binds to Cdt1 and prevents it from loading Mcm complex onto origin DNA.
What is Mcm?
minichromosome maitenance
What is the function of Mcm?
An enzyme which utilizes the energy of ATP hydrolysis to translocate along one strand of the duplex and unwind the complimentary strand.
What is APC/C mediated ubiquitin proteolysis?
It is an E3 ubiquitin ligase that marks target cell cycle proteins for degradation by the 26S proteasome.
What is Cdt1?
Cdt1 is a novel replication factor conserved throughout evolution.
What is cycA?
Cyclin A is the only cyclin that regulates multiple steps of the cell cycle.
It associates with, and thereby activates, two distinct CDKs – CDK2 and CDK1.
Depending on which CDK partner cyclin A binds, the cell will continue through the S phase or it will transition from G 2 to the M phase.
What is CMG?
CMG is the eukaryotic replicative helicase.
CMG contains the ring-shaped hexametric Mcm2–7 that harbours the helicase motors.
What is the GINS complex?
The GINS complex consists of four paralogous subunits.
At the G1/S transition, GINS is recruited to the origins of replication where it assembles with cell-division cycle protein (Cdc)45 and (MCM)2–7 to form the Cdc45/Mcm2–7/GINS (CMG) complex, the presumed replicative helicase.
What is CDC45?
CDC45 is a protein that in humans is encoded by the CDC45L gene.
Outline the DNA unwinding, the formation of the DNA replication bubble with replication forks during initiation?
- Helicase unwinds and separates the double stranded DNA by breaking the hydrogen bonds between base pairs
- Creating a DNA replication bubble with two “Y” shape DNA replication forks of which two polynucleotide strands separated in antiparallel directions
- Single-stranded binding proteins or RPA (replication protein A): Bind to the unwound and separated strands.
*This stabilizes and prevents them do not base-pair with one another again and staying long enough for DNA replication - DNA relaxation at front of the replication fork occurs using Type IIA topoisomerase.
*Making temporary single-stranded “nicks” (Single PDE bond breaks) in one of the two template strands to relieve the torsional stress and supercoiling caused by the unwinding of the helix.
What is type IIA topoisomerase?
Type IIA topoisomerases form double-stranded breaks with four-base pair overhangs.
Outline the stages of elongation.
*Form a DNA/sliding clamp.
*A homotrimer of PCNA protein complex encircling the DNA where it acts as a scaffold,
*This then interacts with DNA polymerase δ or polymerase III to prevent them from dissociating from the DNA template
*This serves as a processivity-promoting factor in DNA replication.
What is PCNA?
Proliferating cell nuclear antigen is a DNA clamp that acts as a processivity factor for DNA polymerase sigma in eukaryotic cells and is essential for replication.
What type of replication does DNA go through?
DNA replication is semi-conservative
What is the end replication problem?
*In human cells, there is no nucleotide to provide the 3’ end –OH for DNA polymerase to fill in the gap when the RNA primer has been removed.
*The single-stranded overhangs produced by incomplete end replication.
*This causes the chromosome shortens significantly with each round of cell division.
How does telomere replication maintain the end of chromosome integrity?
*Have circular DNA molecules as chromosomes.
*Eucaryotes have telomeres at the ends of their chromosomes.
*The telomerase functions as a reverse transcriptase and uses a short RNA template to synthesize the complementary telomere repeats.
*The primase makes the RNA primers using the extended telomere sequence as the template, and the DNA polymerase d & III synthesize the complementary strand to the overhang sequence.
What is a telomere?
A telomere is a region of repetitive DNA sequences at the end of a chromosome. Telomeres protect the ends of chromosomes from becoming frayed or tangled.
Each time a cell divides, the telomeres become slightly shorter.
Eventually, they become so short that the cell can no longer divide successfully, and the cell dies.
The real simple sum up of initiation?
- Origin recognition and activation
- Licensing and assembly of the replication complex
- DNA unwinding, Release superhelical tension, Stabilisation of single-stranded DNA, and the formation of the DNA replication forks
The real simple sum up of elongation?
- Synthesis of a primer for initiation
- Replication polymerase: bidirectional replication; Okazaki fragment
- Leading and lagging stand templates
- Processivity factor: PCNA loader
- Proofreading & Removal of RNA primers
- Ligation of discontinuous DNA fragments
- Semiconserved replication
What is CDE?
cell cycle dependent element
What is CHR?
cell cycle genes homology region
What regulates the G2/M genes transcriptions?
Cyclin B1, CDK1, Aurora kinase and other G2/M genes.
What is the process of Normal G2/M progression?
Antagonized phosphorylation and dephosphorylation of CDK1-cyclin B control G2/M transition.
What are DNA damage checkpoints?
- DNA damage checkpoints are biochemical pathways that delay or arrest cell cycle progression in response to DNA damage.
- Acts as constant surveillance and response systems in that they continuously monitor the integrity of the genome and control cell cycle progression accordingly.
What kind of damage does DNA replication stress cause?
Base mismatches
Insertion and deletion
Which DNA repair mechanism fixes base mismatches and insertion/deletion?
Mismatch repair
What kind of DNA damage is caused by polyaromatic?
DNA adducts pyrimidine dimers intrastrand crosslinks
What kind of DNA damage is caused by hydrocarbons?
DNA adducts pyrimidine dimers intrastrand crosslinks
What kind of DNA damage is caused by UV light?
DNA adducts pyrimidine dimers intrastrand crosslinks
What kind of DNA damage is caused by hydrocarbons?
DNA adducts pyrimidine dimers intrastrand crosslinks.
What DNA repair mechanism are used on DNA adduct pyrimidine dimers instrastrand crosslinks?
Nuclear excision repair
Translesion synthesis
What DNA damage do oxygen radicals cause?
Single strand (ssDNA) break & base oxidation.
What DNA damage does ionizing radiation cause?
*Single strand (ssDNA) break & base oxidation
*Interstrand crosslinks
*Double strand break (DSB)
What DNA damage do chemotherapeutics cause?
*Single strand (ssDNA) break & base oxidation
*Interstrand crosslinks
*Double strand break (DSB)
What DNA repair mechanism works on Single strand (ssDNA) break & base oxidation?
Base excision repair
What DNA repair mechanism works on interstrand crosslinks?
Interstrand crosslinks repair
What are the DNA repair mechanisms used to repair double strand breakage?
Homologous recombination
Alternative Pathway for Non-Homologous End Joining
Non-homologous end-joining
What are the consequences of DNA damage?
-Transient cell cycle arrest
-Mutations chromosome aberrations
-Inhibition of:
* Transcription
* Replication
* Chromosome segregation
What are the stages to the check point cascade?
Sensors
Mediators
Transducers
Effectors
Targets
What are the basic stages of base/nucleotide excision repair?
(1) recognition of damaged DNA.
(2) excision of damaged DNA.
(3) DNA synthesis to fill the nucleotide gap.
(4) sealing of nicks in the DNA.
What are the differences between BER and NER?
BER is able to repair small damages caused endogenously while NER is able to repair damage regions up to 30 base pair length caused mostly by exogenously. BER differs from NER in the types substrates recognized and in the initial cleavage event.
What are the specific differences from the basic stages during base excision repair (BER)?
*It removes the base using a DNA glycosylase.
*Then it removes the tiny bit of sugar phosphate using AP endonuclease and phosphodiesterase.
*DNA polymerase-beta and DNA ligase seals this small nick.
What are the specific differences from the basic stages during nuclear excision repair (NER)?
*It removes the base using a DNA helicase.
*Then it removes the whole sequence of bases using excision nuclease.
*DNA polymerase-beta/ and DNA ligase seals this small nick.
What is Non-Homologous End Joining (NHEJ)?
Non-homologous end joining is an error-prone system for DNA repair that can result in loss of sequence information around the break.
What are the stages of Non-Homologous End Joining (NHEJ)?
Breaking
Sensing/recognition
End processing
Strand invasion, DNA synthesis & resolution.
Ligation
What is Homologous recombination repair (HR)?
Homologous recombination repair (or Homology-directed repair), uses regions of homology from sister chromatids as a template to preserve sequence integrity in response to a double-strand break
What are the stages of Homologous recombination repair (HR)?
*Double strand break.
*Resection
*Exonuclease degrades 5’ ends
*Strand invasion
*D-loop formation
*DNA synthesis & branch migration
*Resolution branch by pairing of newly synthesized DNA with top strand. Top-strand DNA synthesis.
*DNA ligation.
What does diploid mean?
Cells with 2 copies of each chromosome are diploid.
1 chromosome set from mother, and 1 from father.
What needs to happen during mitosis?
*Chromosomes condense.
*Chromosomes attach to spindle microtubules.
*Chromosomes align on the spindle.
*Sister chromatids separated.
*Chromosomes decondense and cell divides into two
What are the stages of mitosis?
Prophase
Prometaphase
Metaphase
Anaphase
Telophase
Cytokinesis
What is entry into mitosis driven by?
M-Cdk (Cyclin B-Cdk1), Known as the “master regulator” of mitosis.
What does M-Cdk (Cyclin B-Cdk1) do?
- Directly phosphorylates key substrate proteins
- regulates downstream mitotic kinases which then phosphorylate additional substrates
What is the Kinetochore?
- The microtubule binding site on a chromosome
- A large macromolecular complex that assembles on the centromere
How are Kinetochore recruited?
M-Cdk (Cyclin B-Cdk1) and Aurora B kinases are required to recruit kinetochore proteins in early mitosis.
What causes chromosomes to condense?
Condensins I and II co-operate to condense chromosomes in mitosis
What are the stages of chromosome condensation?
Cohesin removed
Condensins recruited
Interphase chromosome structure lost, mitotic structure gained
What is prophase?
Interphase chromosome structure is lost
Chromosomes condense
Kinetochore assembly begins
Microtubule dynamics change so that the spindle starts to form
What is prometaphase?
Nuclear envelope breaks down
Microtubules attach to chromosomes
Microtubule adapter proteins and motor proteins become active
Why does the nuclear envelope breaks down?
Allows access of microtubules to chromosomes.
What is the function of microtubule adapter protein and motor proteins?
Allows chromosomes to be moved on the spindle.
What is the mitotic spindle?
A microtubule-based machine required to align and segregate chromosomes.
What is the structure of microtubules?
Nucleated at the minus end
Can grow and shrink at the plus end
(dynamic instability)
What is the function of microtubule adaptor proteins?
*Allow cell components to bind microtubules
*Modulate the stability of microtubules
What is the function of motors?
Allow cell components to move along microtubules
Which side does the motor kinesin-5 tend towards?
Walks to plus ends
Which side does the moto dynein tend towards?
Walks to minus ends.
How do motors move?
Kinesin motors use the energy from ATP to walk along chromosomes.
Can carry various “cargo” proteins
What is kinesin-5?
Forms dimers and cross-links microtubules.
Cross-links anti-parallel microtubules and pushes them apart.
What is the cargo kinesin carries?
Another kinesin-5 molecule.
What is CENP-E?
Binds to kinetochores.
Moves kinetochores towards the cell equator
What is the cargo CENP-E?
A kinetochore
What are the stages of mitotic spindle assembly?
Nucleation of microtubules at centrosomes (minus ends)
Formation of interpolar microtubules and sliding moves centrosomes apart
Nuclear envelope breakdown allows microtubules to capture kinetochores
Which processes require bi-orient on the spindle?
*Making correct attachments (error correction)
*Preventing cell cycle progression until suitable attachments are made (spindle checkpoint)
What is error correction?
*A trial-and-error process
*Only chromosomes that are bi-oriented are stably attached to microtubules
What is the function of aurora B?
Aurora B kinase localizes to centromeres by it detects tension.
Aurora B phosphorylates Ndc80 to remove microtubules from kinetochores.
What is metaphase?
Chromosomes are all bi-oriented which means they align on the “metaphase plate”.
What is the spindle checkpoint?
*If chromosomes are incorrectly attached to the spindle, error correction produces unattached kinetochores
*The spindle checkpoint detects unattached kinetochores
What are the stages of the spindle checkpoint?
Unattached kinetochores produce the Mitotic Checkpoint Complex (MCC).
The MCC inhibits the APC/C and so prevents M-cyclin (Cyclin B) degradation.
This keeps cells in mitosis.
What happens once crisis is avoided at the spindle check point?
Once all kinetochores are occupied with microtubules, the MCC is no longer produced
M-cyclin (Cyclin B) is degraded, and the cells “biochemically” exit mitosis
Numerous mitotic processes are terminated.
What allows the cell to exit mitosis?
Co-ordinated degradation of M-Cyclin and Securin ensures that “biochemical” exit from mitosis and the onset of anaphase chromosome movements occur together.
What happens during anaphase A?
Chromosomes move towards the spindle poles
How do the chromosomes move towards the spindle poles during anaphase?
Driven largely by microtubule depolymerization at the plus ends of kinetochore microtubules (in human cells).
What happens during anaphase B?
Spindle poles move apart.
How do the spindle move apart during anaphase?
Driven largely by microtubule motors eg kinesin-5.
What happens during telophase?
- Nuclear envelope reforms, and nuclear pores inserted
- Chromosomes decondense
How do chromosomes decondense in telophase?
- Condensins dissociate
- Cohesins re-associate and enable the formation of chromosome looping structures needed for correct gene expression
What happens during cytokinesis?
The cell divides into two (cytokinesis)
A contractile ring of actin and myosin drives cleavage furrow formation and pinching of the dividing cell into two.
What signals are involve in cytokenesis?
central spindle
spindle poles
chromosomes
Where is asymmetric cell division used?
- Proteins, RNAs, lipids
- Histone modifications, DNA methylation
- Organelles (old mitochondria)
- Cytoskeletal organization (centrosomes)
- Cell fate components
- Reactive species, protein aggregates
What is asymmetric division used for?
Differentiation
What is symmetric division used for?
Expansion
What does asymmetric divsion start with?
Stem cell.
What are the daughter cells of asymmetric division?
Stem cell
Differentiate
What happens when there are more stem cells than differentiated as a result of asymmetric division?
Cancer
What happens when there are more differentiated cells than stem cells as a result of asymmetric division?
Tissue ageing/degeneration
What are the daughter cells in germ cell asymmetric divsion?
Rejuvenated cell linage which then differentiates into sex cells.
Less fit cell due to senescence factors.
Why do germs use asymmetric division?
Supports their immortality.
What are the steps to asymmetric division?
Interphase
Mitosis
Division
Why is cell polity important for asymmetric division?
Cell polarity refers to the intrinsic asymmetry observed in cells, either in their shape, structure, or organization of cellular components.
This allows for the daughter cells to be different.
How cells transduce cortical polarity to spindle positioning?
Evolutionary conserved machinery involved in mitotic spindle positioning.
What is PAR?
PAR protein affect cell polarity.
anti-PARs and post-PARs are constantly fighting for dominance.
How does PAR affect asymmetric division?
The Par complex directs asymmetric cell division by phosphorylating the cytoskeletal protein.
What is the result of Anterior PAR being depleted?
posteriorized zygote
What is the result of Posterior PAR being depleted?
anteriorized zygote
What are Protooncogenes?
When the cell is forced into the cell cycle
Stimulate aberrant cellular growth and division
What are tumour suppressors?
The cell does not exit the cell when required their loss leads to a defective break.
What are the Chromosomal instabilities in cancer?
Generation of abnormal chromosome numbers (aneuploidy) in mitosis through mis-segregation of chromosomes.
Chromosome rearrangements (translocations)
Deletions
Amplifications,
Insertions
What are the Causes of aneuploidy in mitosis?
- Inappropriate kinetochore-MT attachments
- Compromised SAC (rare in cancer)
- Supernumerary centrosomes
- Problems in chromosome cohesion
- Tetraploidy (4n)
How do cancer cells tolerate aneuploidy?
Mistakes in cell division frequently lead to p53 activation
* p53 is one of the most frequent events in tumorigenesis and allows cancer cells to tolerate a broad range of insults, including chromosome instability (CIN)
