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Genetics > DNA Replication > Flashcards

DNA Replication Flashcards

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1
Q

Semi-conservative DNA replication

A
  • 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
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2
Q

Meselson-Stahl Experiment Key Knowledge

A
  • 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
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3
Q

The Meselson-Stahl Experiment proving semi-conservative replication

A
  • 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)
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4
Q

Meselson-Stahl Experiment - Additional analysis

A
  • 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
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5
Q

Requirements of DNA synthesis

A
  • Template - a region of single stranded DNA
  • All 4 deoxynucleoside triphosphates (dNTPs)
  • A primer - a 3’ OH group that the new nucleotide is added
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6
Q

How DNA polymerase works

A
  • 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

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7
Q

How DNA synthesis is intiated

A
  • 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
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8
Q

Origin of replication - Replication forks

A
  • 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
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9
Q

Termination of DNA replication

A
  • On opposite side to the Ori is termination region - Ter
  • The 2 replication forks will approach ‘ter’ from opposite sides
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10
Q

Leading & Lagging strand synthesis

A
  • 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’)
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11
Q

Replacing the primer & ligation

A
  • 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
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12
Q

Protein factor needed for DNA replication : Initiator protein

A

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

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13
Q

Protein factor needed for DNA replication: Helicase

A

Unwinds the DNA at the replication fork

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14
Q

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

A

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

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15
Q

Protein factor needed for DNA replication: Primase

A

Synthesises the RNA primer

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16
Q

Protein factor needed for DNA replication: DNA polymerases

A
  • DNA polymerase I removes + replaces the RNA primer
  • DNA polymerase III responsible for synthesis of leading + lagging strands
17
Q

Replisome

A
  • Replication requires assembly of ‘large machine’ at replication fork
  • In E.coli, replisome moves along DNA template at 1000 bp per minute
18
Q

Proof-reading by DNA polymerase

A
  • 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
19
Q

How is DNA polymerase a template driven enzyme?

A

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

20
Q

Post-replication repair

A
  • Corrects proof-reading errors
  • Results in error rate of 1 mistake per 10^9 - 10^10 nucleotides added
21
Q

DNA is degraded by nucleases: Exo-

A
  • Cleave nucleotides one at a time from the end of a polynucleotide chain
22
Q

DNA is degraded by nucleases: Endo-

A

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

Genetics (39 decks)

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