Week 4- DNA replication, cell cycle and cancer

Question 1

What is semi-conservative DNA replication

Answer 1

DNA replication is semi-conservative in the next generation molecule where one strand is old and the other strand is new

Question 2

Mechanism of DNA replication

Answer 2

• During DNA replication, upon separation, each DNA strand is used as a template to synthesize the complementary DNA strand
• DNA strand is always synthesized from in the 5’ to 3’ direction
• phosphodiester bonds
• Complementary strands bring in individual nucleotides to base pair with the complementary bases on the template
• there is no restriction replication as opposed to transcription
• as a result, the whole DNA genome is duplicated

Question 3

Basic DNA replication

Answer 3

• Single replication origin ( multiple in eukaryotes)
• RNA primer (synthesized by RNA polymerase called primase)
• DNA polymerase
• Replication fork (continuous and discontinuous DNA replication at leading and lagging strands respectively)

Question 4

What are DNA polymerase

Answer 4

• the enzyme that synthesizes DNA polymer
• Requires RNA primer for DNA synthesis to begin
• Achieved by forming a bond between 5’ phosphate of incoming nucleotide and 3’ OH of end of growing strand: DNA synthesis in 5’ to 3’ direction

Question 5

RNA primers at the lagging template

Answer 5

1. New RNA primer synthesis by primase
2. DNA polymerase adds to new RNA primer to start DNA fragment
3. DNA polymerase finishes DNA fragment
4. Old RNA primer erased and replaced by DNA
5. Nick sealing of DNA ligase joins new DNA fragments to the growing chain

Question 6

Control of Cell Cycle: checkpoints

Answer 6

• Surveillance mechanisms to ensure - no DNA damage and completion of DNA replication
• Activated throughout cell cycle
• Detection of defect leads to arrest of cell cycle progression, until defect is repaired
• Failure at checkpoints usually leads to cancer development

Question 7

Example of checkpoints

Answer 7

• ATM coded for kinase activated by DNA damage
• ATM phosphorylates and activates Chk 2 (a checkpoint protein)
• Chk 2 phosphorylates p53
• Phosphorylated p53 activates gene expression of p21
• Cells with DNA damage in G1 synthesize p21 protein
• p21 inhibits kinase activity of G1 cdk
• The inhibition prevents cells from entering S phase
• Thus, mutated p53 found in cancer may be the cause of failure in checkpoint control and subsequent development of cancer

Question 8

Basic properties of cancer cells

Answer 8

Loss of growth control
- Normal cells show contact inhibition
- Cancer cells continue to grow in confluency and form colonies
- Normal cells respond to growth factors
- Cancer cells do not respond to such signals
- Normal cells have limited cell cycles
- Cancer cells are immortal

Question 9

Tumor-Suppressor pRB and Cell cycle

Answer 9

• pRb helps to regulate G1 to S phase transition, and helps to commit the cell to divide
• G1-S transition requires activation of many genes (polymerases, cyclins)
• pRb forms complex with E2F (transcription factor of cell cycle genes), and prevents E2F’s function
• Unphosphorylated E2F-pRb complexes arrests cell cycle at G1, phosphorylation of pRb by cdk releases E2F and allows cell cycle to resume
• Mutations in RB remove its inhibitory effects on E2F

Question 10

p53 and Cell cycle

Answer 10

• Cells with DNA damage in G1 synthesize p21 protein with higher levels of p53
• p21 inhibits kinase activity of G1 cdk (which drives cell through G1)
• arrest at G1 allows cells to repair DNA damage
• Mutation in both copies of TP53 lead to absence of p21
• Absence of p21 allows cells to proceed with cell cycle without repair of DNA damage
• Result: higher risk of cancer