DNA Replication

Question 1

When in the cell cycle does DNA replication occur and how long does it last?

Answer 1

S phase

8 hrs

Question 2

What are replication forks?

Answer 2

Y shaped structures where new DNA strands are synthesised by the replicosome (multi-enzyme complex).

Question 3

Where does DNA replication begin?

Answer 3

At origins of replication - 1 in prokaryotes, multiple in eukaryotic chromosomes due to greater size of genome.

Question 4

Describe the steps in DNA replication?

Answer 4

1. Helicases unwind the parental double helix.
2. Single-strand binding proteins stabilise the unwound parental DNA.
3. The leading strand is synthesised continuously in the 5’-3’ direction by DNA polymerase.
4. The lagging strand is synthesised discontinuously: primase synthesises a short RNA primer which is extended by DNA polymerase to form an Okazaki fragment.
5. After the RNA primer is replaced by DNA (by another DNA polymerase), DNA ligase joins the Okazaki fragment to the growing strand.

Question 5

What is the DNA replication reaction driven by?

Answer 5

Pyrophosphate hydrolysis (to add a dNTP to the growing DNA strand)

Question 6

What are DNA polymerases?

Answer 6

Enzymes that create DNA molecules by assembling nucleotides in a complementary way to a template DNA strand.

Question 7

In which direction does DPol synthesise DNA and read the template DNA?

Answer 7

• Reads template strand in 3’-5’ direction.

- Assembles new DNA strand in 5’-3’ direction.

Question 8

What does DNA polymerisation require to begin?

Answer 8

A pre-existing RNA primer from which to extend.

Question 9

How does DPol reduce its replication error rate?

Answer 9

• Contains an exonuclease domain: detects base pair mismatches and removes the incorrect nucleotide (to be replaced by the correct one).
• Reduces error rate from 1/10^6 to 1/10^9.

Question 10

What can be the result of mutations in the DPol exonuclease domain?

Answer 10

• Tumours

- E.g. Ultramutated group of endometrial carcinomas characterised by mutations in DPolE exonuclease domain.

Question 11

What are endonucleases and exonucleases?

Answer 11

Both are nuclease enzymes that catalyse the hydrolysis of single nucleotides present in a DNA chain at specific sequences:

• exonucleases cleave nucleotides at the ends of the DNA molecule (requires free 3’ or 5’ hydroxyl group)
• endonucleases cleave bonds within the molecule

Question 12

What is DNA replication stress?

Answer 12

Inefficient replication that leads to fork slowing, stalling and/or breakage.

Question 13

What are the consequences of errors in DNA replication?

Answer 13

Can lead to genomic instability - can cause either mutations or cell death depending on context - both can cause disease.

Question 14

Describe the factors causing DNA replication stress.

Answer 14

1. Defect in replication machinery (e.g. Helicase stalling)
2. Factors hindering replication fork progress, e.g.
   
   • DNA lesions
   • DNA secondary structure
   • Repetitive DNA
   • Fragile sites or oncogene-induced stress
3. Defects in the response pathways to the above factors, e.g.
   
   • DNA repair pathways
   • Regulation of origin firing

Question 15

How does splippage of the replication machinery cause errors?

Answer 15

• Slippages occur at trinucleotide repeats - microsatellites - due to repetitive nature of sequence.
• 2 scenarios:
  
  • newly synthesised strand loops out - 1 nucleotide is added on the new strand - extra nucleotide.
  • template strand loops out - 1 nucleotide is omitted on the new strand - missing nucleotide.

Question 16

What is the result of defects in the exonuclease activity (3’-5’) of DPol?

Answer 16

Increased mutation rate as mismatches aren’t removed.

Question 17

Which defects allow the persistence of DNA strand breaks?

Answer 17

• Base excision repair (BER) defect causes DNA single-strand breaks to persist.
• BRCA deficiency causes DNA double-strand breaks to persist, e.g. In some forms of breast and ovarian cancer.

Question 18

Why are DSBs the most ‘dangerous’ types of DNA damage?

Answer 18

• Disrupt molecular continuity
• Difficult to resolve
• Can lead to mutations or cell death if not repaired correctly. Mutations include translocations and chromothrypsis (chromosomal rearrangements)

Question 19

Which hypothesis explains why age is the biggest risk factor in cancer?

Answer 19

Mutation accumulation hypothesis

Question 20

Which disease is associated with a mutation in helicase?

Answer 20

• Werner protein is a helicase important during DNA replication; defects lead to DNA replication faults and DNA damage.
• WERNER SYNDROME involves autosomal recessive WRN gene mutations.
• Symptoms: premature display of features associated with normal ageing, inc. cataracts, skin ulcers, type 2 diabetes, atherosclerosis, osteoporosis and cancer predisposition.

Question 21

Suggests endogenous and exogenous sources of DNA damage.

Answer 21

Endogenous:

• DNA replication errors
• V(D)J recombination
• Free radicals

Exogenous:

• Ionising radiation
• Alkylating agents
• UV light
• Anti-cancer agents

Question 22

What is the rate of DNA damage from environment and normal metabolism?

Answer 22

Up to 1 x 10^6 molecular lesions/cell/day.

Question 23

Suggest examples of DNA damage and their causes.

Answer 23

• Apurinic/apyrimidinic site: spontaneous DNA loss due to label nature of N-glycosidic bond linking nucleobase and pentose sugar.
• Deamination (loss of amine group).
• Mismatches: replication error.
• Single strand breaks
• Double stand breaks: free radicals generated as a byproduct of metabolism or exposure to exogenous physical agents (e.g. Ionising radiation)
• Pyrimidine dimer: non-ionising UV radiation
• Bulky adducts or inter- & intra-strand crosslinks: chemical compounds (e.g. Cisplatin): block DNA replication