DNA replication

Question 1

Describe Meselson & Stahl’s 1958 experiment into DNA replication

Answer 1

1. Grow bacteria in media containing 15N = ‘heavy’ DNA
2. Transfer them to media containing 14N = ‘light’ DNA
3. Separate heavy and light molecules by ultracentrifugation → caesium chloride gradient
4. Heavy DNA sunk towards the bottom of the tube while lighter stays closer to the stop
5. Visualise using UV light

Question 2

What did Meselson & Stahl’s 1958 experiment demonstrate about DNA replication?

Answer 2

That it is semi-conservative

Question 3

Describe the role of DNA polymerase

Answer 3

• Add nucleotides one at a time, in a 5’-3’ direction (always added to the 3’ end!)
• Using template strand to form H-bonds (how it tells which base to add next)
• 10s or 100s of nucleotides per sec
• Has to have a nucleotide to add on to (primer)

Question 4

Which direction does DNA polymerase work?

Answer 4

5’-3’ direction - nueclotide added to 3’ end

Question 5

Describe the role of primase

Answer 5

Generates the primer (made of RNA) thats built on by the DNA
RNA primer is removed

Question 6

Describe the role of ligase

Answer 6

• Joins loose ends together into a single strand of DNA
• There’s base pairing between nucleobases but gaps in the sugar-phosphate backbone that need to be joined

Question 7

Describe the role of helicase

Answer 7

Breaks H-bonds between the 2 DNA strands, separating them

Question 8

Describe the role of single strand binding protein (SSB)

Answer 8

Binds to separated strands, preventing them from reannealing

Question 9

Describe the role of Topoisomerase in DNA replication

Answer 9

Relieves pressure from overwinding around the replication bubble by making and resealing breaks in the DNA

Question 10

Describe the leading strand in DNA replication

Answer 10

5’-3’ DNA synthesis points towards the replication fork and can proceed continuously

Question 11

Describe the lagging strand in DNA replication

Answer 11

5’-3’ DNA synthesis points away from the replication fork and must be discontinuous (multiple primers)

Question 12

What are okazaki fragments?

Answer 12

Pieces of DNA that are stuck together to make up the lagging
strand of replication

Question 13

Describe the erosion of genetic material at the ends of linear chromosomes

Answer 13

• Primer removal at the end of the chromosome leaves a gap that can’t be filled (wrong direction for - DNA polymerase to work)
• Theres an overhand with no base pair
• On every round of replication a small piece is lost from the end of the chromosome
• Problem for the lagging strand

Question 14

Describe telomeres in DNA replication

Answer 14

→ short DNA sequences that are repeated over and over at the end of chromosomes
(Short stretches are lost from telomeres at each round of replication)
- They protect the ends of DNA from eroding

Question 15

What is the role of telomerase in DNA replication?

Answer 15

enzyme that replenishes telomeres from an RNA template
- Repetitive nature of telomeres also allows them to bind to specific proteins, protecting the vulnerable ends of the chromosome.

Question 16

Describe the Shelterin complex

Answer 16

No. of specialised proteins bind telomere DNA → form a cap on the telomere