DNA Replication

Question 1

Semi-conservative DNA replication

Answer 1

• Each strand is a template for synthesis of complementary strands after being unzipped
• Each new double-stranded would have 1 original parent strand + newly made daughter

Question 2

Meselson-Stahl Experiment Key Knowledge

Answer 2

• Bacteria can be grown in a medium containing N-15 or N-14 so DNA contains either N sources
• Equilibrium density centrifugation can be used to separate more dense N-15 DNA from N-14
• Centrifuged cesium chloride
  forming a gradient of density
• DNA is then added and will form a layer/band in the cesium gradient - can determine weight

Question 3

The Meselson-Stahl Experiment proving semi-conservative replication

Answer 3

• Parent DNA labeled with N-15 (grown in15^NH4Cl) for multiple generations - all have heavy N
• DNA was isolated and added to a medium containing 14^NH4Cl
• Grew for 1 generation - formed a lighter hybrid when sampled
• Grew for 2nd generation - had 1 band of light DNA (N-14) and 1 hybrid band (N-15/N-14)

Question 4

Meselson-Stahl Experiment - Additional analysis

Answer 4

• After 1 generation, a sample of DNA was heat-denatured before being analysed by centrifigation
• Forms 2 bands - 1 of N-14 and 1 of N-15 - as DNA strands are split into 2
• Shows that replicated DNA contain 1 strand of parent that’s intact with only N-15 then 1 that’s new with N-14

Question 5

Requirements of DNA synthesis

Answer 5

• Template - a region of single stranded DNA
• All 4 deoxynucleoside triphosphates (dNTPs)
• A primer - a 3’ OH group that the new nucleotide is added

Question 6

How DNA polymerase works

Answer 6

• Incoming dNTP pairs with the base on template strand
• Phosphodiester bond formed and pyrophosphate (2 phosphate groups are released)

DNA polymerase only proceeds in a 5’ to 3’ direction
- Is processive - remains bound/attached to template DNA - if it comes off, replication stops

Question 7

How DNA synthesis is intiated

Answer 7

• DNA polymerase doesn’t synthesise de novo (from nothing)
• Requires a small RNA primer (8-12 bases) synthesized by the primase
• DNA polymerase can then extend the chain

Question 8

Origin of replication - Replication forks

Answer 8

• DNA synthesis starts at a specific point on chromosome at the Ori
• Local melting of DNA at Ori + assembly of 2 replisomes
• Rs then move away from Ori in opposite direction creating bidirectional replication forks
• 2 strands then synthesised at each replication fork

Question 9

Termination of DNA replication

Answer 9

• On opposite side to the Ori is termination region - Ter
• The 2 replication forks will approach ‘ter’ from opposite sides

Question 10

Leading & Lagging strand synthesis

Answer 10

• During DNA replication, the leading is 5’ to 3’ + lagging strand is 3’ to 5’
• Lagging strand is synthesised in small segments - Okazaki fragments that are 1000 - 2000 bases long (synthesised 5’ to 3’)

Question 11

Replacing the primer & ligation

Answer 11

• Lagging strand - the RNA primer will be removed by 5’-3’ exonuclease activity of DNA polymerase then replace missing bases
• ‘Nicks’ between Okazaki fragmets are joined by DNA ligase using ATP, releasing AMP + pyrophosphate

Question 12

Protein factor needed for DNA replication : Initiator protein

Answer 12

Binds ‘Ori’ and unwinds the DNA at the ‘Ori’

Question 13

Protein factor needed for DNA replication: Helicase

Answer 13

Unwinds the DNA at the replication fork

Question 14

Protein factor needed for DNA replication: Topoisomerases (including gyrase)

Answer 14

Relaxes the DNA and stop unwinding of the supercoiled DNA ahead of the replication fork

Question 15

Protein factor needed for DNA replication: Primase

Answer 15

Synthesises the RNA primer

Question 16

Protein factor needed for DNA replication: DNA polymerases

Answer 16

• DNA polymerase I removes + replaces the RNA primer
• DNA polymerase III responsible for synthesis of leading + lagging strands

Question 17

Replisome

Answer 17

• Replication requires assembly of ‘large machine’ at replication fork
• In E.coli, replisome moves along DNA template at 1000 bp per minute

Question 18

Proof-reading by DNA polymerase

Answer 18

• If an incorrect base is added, DNA polymerase has a 3’-5’ exonuclease which removes the mismatched nucleotide
• Results in an error rate of 1 mistake per 10^7 bases copied

Question 19

How is DNA polymerase a template driven enzyme?

Answer 19

A phosphodiester bond will only be formed if the incoming nucleotide pairs with the base on the template

Question 20

Post-replication repair

Answer 20

• Corrects proof-reading errors
• Results in error rate of 1 mistake per 10^9 - 10^10 nucleotides added

Question 21

DNA is degraded by nucleases: Exo-

Answer 21

• Cleave nucleotides one at a time from the end of a polynucleotide chain

Question 22

DNA is degraded by nucleases: Endo-

Answer 22

Cleave bonds within a DNA chain e.g. DNAse
- Restriction endo- recognise specific sequences + then cleave the DNA