Cell Cycle

Question 1

In cell cycle eukaryotes…

Answer 1

Replace lost body cells

Replace old and worn out cells

Undergo clonal expansion

Embryonic development

Question 2

Interphase

Answer 2

G1, S, G2 sub-phases
(G- gap (growth) between main phases)
(S- synthesis of DNA)

Question 3

M phase

Answer 3

Mitotic phase

• Mitosis: division of nucleus
• Cytokinesis: division of cytoplasm and organelle

Question 4

Cells out of cell cycle are what?

Answer 4

G0 (G zero)

Question 5

What cells lose their ability to enter cell cycle?

Answer 5

Post-mitotic cells: terminally differentiated cells that have lost their
ability to replicate.

• They are said to be permanently arrested.
• Usually highly specialised cells.
• Contact inhibition can also cause cells to exit the cell cycle

Question 6

What cells can enter the cell cycle?

Answer 6

Cells with high mitotic activity
• High turnover cells are constantly in the cell cycle.

Cells that divide upon appropriate stimulation
• Most of the cells in our body only divide upon stimulation from growth
factors, hormones or other signals.

Question 7

G1 phase

Answer 7

• Stimulus or signal (eg growth factor) required for entry from G0.
• G1 is the longest phase of cell cycle & cell volume doubles

• Period where the cell grows and prepares for S phase.
(e.g metabolic changes preparation for S phase ensures adequate reserves for organelles to replicate, Synthesis of enzymes and other cellular components needed for DNA synthesis)

• Entry to S phase will be delayed if the cell is not fully prepared

• In unfavourable conditions such as growth deprivation or in the presences of growth inhibitory signals the cell can withdraw from G1 and go back to G0 until conditions improve
• Many biological processes in this phase are driven by regulatory proteins specific to G1

Question 8

S phase

Answer 8

• Replication is semi-conservative
• Original DNA strands used as templates for the synthesis of new strands.
• Daughter cells inherit one parental DNA strand and one new strand.
• Occurs in 3 steps
• Initiation > Elongation > Termination

Question 9

DNA replication: INITIATION

Answer 9

DNA UNWINDS
• DNA Helicases are necessary for this. Process requires ATP to
the break hydrogen bonds between base pairs

UNWINDING = SUPERCOILING IN OTHER REGIONS OF DNA
• DNA topoisomerases relieve this tension by unwinding
‘supercoiled’ regions in the DNA, they also prevent knotting
downstream of the DNA so that Helicase can continue strand
separation
• Topoisomerases work by cutting one or both strands of the
DNA duplex at key points, untwisting the strands to relieve the
tension, then resealing the breaks.

UNWOUND DNA KEPT OPEN FOR REPLICATION
• Single strand binding proteins (SSB) bind temporarily to
separated DNA strands as soon as they have been unwound by
DNA Helicase to keep the strands apart during replication

Question 10

Origins of Replication

Answer 10

• The size & complexity of the genome necessitate multiple replication starting points called Origins of Replication (ORC) for rapid DNA synthesis
• ORC occur along the length of each chromosome, In humans approx 30,000 to 50,000 ORC are visible along the length of chromosomes during S phase

Question 11

Helicases unwind DNA in what way?

Answer 11

Helicases unwind DNA away from the origin of replication in a bidirectional manner. This creates replication bubbles that grow bigger in both directions as DNA unwinding progresses

Question 12

Replication fork

Answer 12

The Y-shaped region at the furthest end of each replication bubble is referred to as the replication fork.

Question 13

DNA replication: ELONGATION

Answer 13

• In humans, DNA Polymerase  (delta) is the key player in this step. However it cannot synthesise DNA de novo so a primer is required
• Primases (DNA Polymerase ) short RNA primers, which serve as starting points for the DNA elongation process. Primers can be 10-20 primers long and provide the initial hydroxyl group for elongation
• DNA polymerase  then sequentially adds DNA nucleotide in the 5’ to 3’ direction of the DNA strand.
• The DNA double helix is anti-parallel. One strand runs in the 5’ to 3 direction. The other strand runs in the 3’to 5’
• DNA replication is therefore bi-directional

Question 14

Leading strand

Answer 14

5-3 direction, replication is continuous in elongation

Question 15

Lagging strand

Answer 15

Anti-parallel strand
in the opposite direction to the leading strand but in a fragmented manner (in keeping with the 5’ to 3’ extension rule)

The fragmented DNA strands are known as Okazaki fragments

Question 16

DNA replication: TERMINATION

Answer 16

• An endonuclease removes the RNA primer
• DNA polymerase fills the gaps with DNA nucleotides according to the rule of base pairing
• DNA ligase joins the Okazaki fragments to produce a continuous chain
• DNA polymerase can proofread its own work. If mistakes happen it removes them immediately.
• Other DNA repair mechanisms can be activated to correct errors as necessary

TELOMERES
• Removal of last RNA primer at the very end of lagging strand creates a gap which cannot be filled since DNA polymerase works from a primer template.

• Telomerase enzymes correct this shortfall by adding non coding 6-8 bp DNA repeats
to the end fragments.

• These repeat sequences of non coding DNA at chromosomal ends are called telomeres. They ‘cap’ (stabilise) chromosome ends and prevent loss of genetic information. They also prevent the ends from becoming tangled.
• Telomeres progressively shorten (senescence) in certain types of cells. When telomere length shortens to a critical point the cell dies.

Question 17

DNA replication vs PCR

Answer 17

PCR:

• No Helicase, Topoisomerase or Primase necessary
• DNA primers (not RNA)
• No leading or lagging strands

Question 18

S phase: DNA REPLICATION SUMMARY

Answer 18

Question 19

DNA REPAIR MECHANISMS FOR

1. Single strand DNA damage
2. Double strand DNA damage

Answer 19

Single:
Base excision repair
Nucleotide excision repair
Mismatch repair

Double:
Non-homologous end joining
Homologous recombination repair

Question 20

DNA repair failure in human cancers

Answer 20

BRCA1 and BRA2 defects due to double strand breaks- cannot be repaired so join together by non-homologous recombination so lose a piece of DNA in breast cancer

Question 21

What happens at end of S phase?

Answer 21

Chromosomal DNA content doubles (2N to 4N)
• Two identical strands of each chromosome.
• Sister chromatids joined at the centromere.

moves on to G2 phase

Question 22

G2 phase

Answer 22

• Generally shorter than G1.
• Cell grows and prepares for mitosis.
• Synthesis of cellular components for mitosis (proteins and RNA synthesis) e g organelles such asthe centrioles and mitotic spindle proteins.
• Many biological processes in this phase are driven by regulatory proteins specific to G2.
• The cell will delay entry into M phase to correct errors that will affect the mitosis. eg DNA repair
• Apoptosis will occur if the DNA is damaged and cannot be repaired

then onto M phase

Question 23

M phase

Answer 23

MITOSIS (PMAT):
• Division of nucleus.
• Involves 4 main phases, based on the physical state of the
chromosomes and spindle.
• Prophase, Metaphase, Anaphase & Telophase

CYTOKINESIS
• Final step in the cell cycle.
• Division of cytoplasm and organelles

Question 24

MITOSIS STEPS

Answer 24

• Prophase: chromosomes condense, centrosomes move to opposite poles, mitotic spindle forms
• Metaphase: centrosome are at opposite poles, chromosomes are at their most condensed and line up at the equator of the mitotic spindle. Mitosis will not continue if chromosomes are not properly aligned

• Anaphase: sister chromatids separate
synchronously, each new daughter chromosome moving to the opposite spindle pole.

• Telophase: chromosome arrives at the spindle poles, chromosomes decondense, nuclear envelope reforms

Question 25

CYTOKINESIS

Answer 25

Division of cytoplasm into identical daughter cells.

Question 26

Meiosis vs Mitosis

Answer 26

Question 27

What happens at the end of M phase?

Answer 27

• Some cells will re-enter the cell cycle at G1 and divide again until senescence (associated with a loss of telomerase activity). • Some cells will stop dividing and arrest in G0 in response to inhibitory signals e.g. contact with neighbouring cells (contact inhibition). • Some cells exit and enter G0 in order to differentiate into specialised cells.

Question 28

What are cell cycle checkpoints and what do they do?

Answer 28

Critical Control point allows cells to • stop cell cycle progression. • correct detected errors before proceeding to the next cell cycle phase. • withdraw from the cell cycle if conditions are not right. • undergo programmed cell death (apoptosis) if errors cannot be corrected. * Ensures DNA replication is done with high fidelity and that the integrity is preserved in daughter cells. * Ensures progression through the cell cycle occurs in the correct sequence. * Involves Checkpoint proteins.

Question 29

What are the 3 major cell cycle checkpoints?

Answer 29

* G1 or G1/S checkpoint: Controls progression from G1 to the S phase. * G2 or G2/M checkpoint: Controls progression from the G2 phase to the Μ phase. * Metaphase (M) checkpoint: Controls progression from Metaphase to Anaphase.

Question 30

Key points on cell cycle regulation

Answer 30

Cell cycle must proceed in a precise order_G1-S-G2-M. * Must occur only once per cell division. * Entry into each phase of the cell cycle is strictly controlled by builtin checks points. * A key mechanism in this process is Phosphorylation and Dephosphorylation. * Process is controlled by sequential expression, activation, inactivation and degradation of phase specific cell cycle regulatory proteins Important ones Cyclins & Cyclin dependent kinases (cdks)

Question 31

What is phosphorylation?

Answer 31

* Addition of a Phosphate molecule to a protein. * Most common post-translational protein modifications in eukaryotic cells. Effects: The strong negative charge on a phosphate group can: • Alter the physical properties modification of the protein shape. • Alter the biochemical e.g how it interacts with water (a hydrophobic protein can will become hydrophilic when phosphorylated. • Result in acute and reversible regulation of protein function e.g modulating protein folding, substrate affinity, stability and activity. Advantages as a control mechanism • It is rapid, taking as little as a few seconds. • It does not require new proteins to be made or degraded. • It is easily reversible. • Particularly important regulation mechanism in the control of cell cycle progression

Question 32

What are cyclins?

Answer 32

* Family of proteins that control the progression of cells through the cell cycle undergo a constant cycle of synthesis and degradation during cell division. * Their concentrations is tightly regulated to ensure the cell cycle progresses in a proper sequence. * Levels and type expressed vary depending on the cell cycle phase. * At their peak levels when the protein they need to regulate is need.

Question 33

What are cyclin-dependant protein kinases?

Answer 33

* Enzyme that adds negatively charged phosphate groups to other molecules by Phosphorylation * Levels fairly stable throughout the cell cycle. * Inactive until a Cyclin binds to it. * Degradation of the bound Cyclin terminates the Cdk activity.

Question 34

What is the cyclin-CDK complex?

Answer 34

* The active complex sole function is Phosphorylation of proteins in a cell cycle phase specific manner. * The phosphorylated protein will then trigger specific events within the cell cycle (e.g. DNA polymerase gene expression (G1) through interaction with Retinoblastoma (Rb) protein. * After the event, the bound cyclin is degraded and the CDK becomes inactive again

Question 35

Phosphorylation of what allows transcription of more Cyclin E?

Answer 35

Rb-E2F complex ``` Retinoblastoma protein (RB): • A key cyclin-CDK target protein in G1 and G1 to S progression. • Transcription Regulator (Gatekeeper). • A major tumour suppressor protein. • can prevent entry into S phase and induce cell-cycle arrest ``` Elongation factor-2 (E2F) • Transcription factors that regulate the expression of genes important in cell proliferation, particularly those involved in progression through G1 and into the S-phase of the cell

Question 36

What families of CKIs are key players in stopping cyclin-CDK activity during cell cycle?

Answer 36

Question 37

p53 in cell cycle

Answer 37

* Activated in response to DNA Damage e.g Radiation by Phosphorylation. * Phosphorylated p53 is active form and has a longer half life. * Induces for CDKI expression (e.g p21) which causes the cell to arrest in G1 until the damaged DNA is repaired. * If the DNA cannot be repaired it induces APOPTOSIS (programmed cell death). * Important tumour suppressor protein.

Question 38

What are mitogens and how do they regulate the cell cycle?

Answer 38

* Cytokines (small proteins 5-80 kDa) that stimulate cell proliferation. * Bind to their cell-surface receptors and activate the signalling pathways that lead to cell cycle initiation and ultimately cell proliferation. • Differentiated cells enter from G0 to G1 after the action of growth factors. • Some mitogen genes and mitogen receptor genes are proto-oncogenes (genes which can mutate to become oncogenes/cancer promoting genes)

Question 39

Cancer and the cell cycle

Answer 39

Cancer starts with a single cell that loses its ability to respond appropriately to growth signals and cell cycle control mechanisms. Cancer Cells: • Ignore normal cell cycle checkpoint signals. • Enter S phase and replicates even if there is genetic damage, ultimately new cancer cells become progressively more abnormal. • Ignore the M checkpoint. Dividing cells containing abnormal chromosomal content produce even more genetically abnormal clones as the disease progresses. • Do not respond to growth inhibitory signals such as contact inhibition. The dividing cells pile on top of each other forming a mass/lump that gets bigger with every round of cell division, suffocating surrounding cells in the process. • Do not respond to appropriate apoptosis signals. They divide continuously (immortal) draining all the body’s resources at the expense of other cells. • Avoid senescence or cell death by maintaining their telomeres despite repeated cell divisions. • Many genes that regulate cell cycle (e.g. p53 and pRB) are mutated in human cancers

Question 40

5 FLUORO-URACIL (5- FU)

Answer 40

S phase active cancer drug An antimetabolite inhibits thymidylate synthase which prevents the synthesis of the nucleoside thymidine (dTMP) required for DNA replication => death of rapidly proliferating cells.

Question 41

S phase active cancer drugs: antibiotics

Answer 41

ANTIBIOTICS DOXORUBICIN & DAUNORUBICIN – Inhibit topoisomerase II & generate free radicals causing DNA damage

Question 42

M phase active cancer drugs

Answer 42

• Colchicine: an agent that stabilizes free tubulin, preventing microtubule polymerization and arresting cells in mitosis – used in karyotype analysis. • Vinca alkaloids: a set of anti-mitotic and anti-microtubule alkaloid agents (e.g. vinblastine, vincristine). They block beta tubulin polymerization and prevent the formation of microtubules and therefore inhibit cell division. They are used in cancer chemotherapy. • Paclitaxel (Taxol): stabilizes microtubules, preventing de-polymerization approved in the UK for ovarian, breast and lung, bladder, prostate, melanoma, oesophageal, and other types of solid tumour cancers as well as Kaposi’s sarcoma.