Replication of DNA

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  • Created by: Jenny Le
  • Created on: 15-04-14 13:41

Mechanisms for DNA replication

There are three different hypotheses for the mechanism of DNA replication:

  • Conservative
  • Semi-conservative
  • Dispersive
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Taylor, Wood and Hughs

Taylor , Wood and Hughs did some early work on DNA replication, they used V.faba roots and H-dTTP.

This shed some light on the problem but it was not until Meselson and Stahl (1958) published their work with E.coli that the problem was solved.

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Meselson and Stahl (1958)

Experiments with E.coli

  • Cultured E.coli in brother containing 15NH4Cl
  • Switched the broth to 14NH4Cl
  • 14N-DNA vs 15N-DNA
  • If the DNA samples are run on CsCl density gradient, 14N-DNA can be distinguished from 15N-DNA as they have different densities. 15N-DNA is heavier than 14N-DNA.

Starting with E.coli grown in brother containing 15NH4Cl (all dNA heavy):

  • Switch broth to 14NH4Cl and take samples at 20 minute intervals
  • 20 minutes = 1 life cycle = 1 round of DNA replication
  • As the DNA replicates, the newly synthesised DNA will be 'light'
  • Run DNA samples on CsCl and can distinguish between the three potential methods of DNA replication

Conclusion:- DNA replication is semi-conservative

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Which enzyme is involved?

Kornberg et al (1957)

  • worked with E.coli
  • isolated DNA polymerase
  • enzymology was not what was expected
  • DNA polymerase was equally good as a nuclease as a polymerase

Cairns and De Lucia (1969)

  • isolated E.coli mutant with only 1% of Kornberg's DNA polymerase activity but which was still able to divide effectively 
  • However it could repair DNA damaged by UV light

DNA polymerase I: a repair enzyme

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DNA Polymerases

There have been two other E.coli DNA polymerases found since the first one:

  • DNA polymerase II
  • DNA polymerase III

They only function 5' to 3' and will not work on ssDNA as they require a short dsDNA region to act as a primer

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Eukaryotic DNA Polymerase

  • DNA replication takes place in the S phase
  • There are give different DNA polymerases found mammalian cells:
    • A (alpha) - replication of nuclear DNA
    • B (beta) - DNA repair
    • Y (gamma) - replication of mitochondrial DNA
    • O (delta) - replication of nuclear DNA
    • E (epsilon) - repair?

Mechanism for DNA replication

Mechanism described is for prokaryotes (E.coli) but similar mechanism is seen in eukaryotes.


  • triphosphate deoxynucleotides dATP, dTTP, dCTP, dGTP
  • DNA template + primer
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Origin of Replication

  • specific DNA sequences
  • 1 per plasmid/bacterial genome
    • eukaryotes have larger genomes so 1 per chromosome would be too slow
    • several per chromosome
    • yeast: 1 every 40kb
    • mammals: 1 every 150kb
  • seen in viral genomes
  • REPLICON: any piece of DNA that replicates as a single unit

At replication, the sequence at the origin becomes single stranded. Once the origin is opened, DNA synthesis is bi-directional.

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DNA synthesis from two origins

  • Starting point - parent DNA strands and an origin of replication identified.
  • Origin is open.
  • DNA synthesis starts to generate daughter strands.
  • Another origin of replication is identified.
  • DNA synthesis has progressed at both origins of replication
  • DNA replication that started at one origin of replication has met that progressing from the other origin. 
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Method for replication of DNA (E.coli)

  • Both strands of DNA are synthesised simultaneously at each replication fork
  • But the replication has to be 5' to 3' 
  • Since strands are antiparallel, how is the strand that runs 5'-3' past the replication fork copied?

Okazaki et al (1968)

  • E.coli +  3H-dTTP incubated for a few seconds
  • extracted DNA
    • newly synthesised DNA will have incorporated the 3H-dTTP
    • isolated small single stranded 3H-DNA fragments ~1000-2000 nt
  • If the experiment was extended
  • E.coli + pulse 3H-dTTP then dTTP
    • the 3H-small fragments are incorporated into high MW DNA

These fragmens were called OKAZAKI FRAGMENTS

  • prokaryotes 1000-2000 nt
  • eukaryotes 100-200 nt
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Semi-Discontinuous Replication Model

  • Leading strand 5' to 3' continuously synthesised
  • Lagging strand5' to 3' discontinuously synthesised
  • Series of Okazaki fragments joined together later

Problems still outstanding:

  • how the primer is set up
  • how to deal with supercoiling/relaxed state as necessary
  • how to maintain ssDNA as required
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Range of Enzymes and Proteins

  • single stranded binding proteins: SSBs
    • bind to ** bubble at the origin of replication
  • helicase
    • breaks the H bonds between the bps. ATP mediated
    • migrates along the parental DNA molecule with the replication fork
  • DNA gyrase (topoisomerase II)
    • mediates unwinding of the dNA in advance of the replication fork
  • primosome (RNA polymerase complex) + rNTP
    • displaces SSBs and catalysed formation of RNA primer complementary to the DNA
    • about 6 nt long
  • replicase (DNA polymerase III) + dNTP
    • replaces primosome, uses RNA as primer
  • DNA polymerase I
    • removes RNA primer and fills in the gap
  • DNA ligase
    • joins together any nucleotides as nece**ary
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Primer production and Leading Strand Synthesis*

  • DNA duplex opened at the origin of replication
  • Helicase and DNA gyrase activity
  • SSBs keep ssDNA as single strand
  • Primosome (RNA polymerase) bind, synthessis complementary RNA molecule = primer
  • SSBs displace
  • Further unwinding of parental duplex at replication fork
  • Primosome replaced by replicase = DNA polymerase III
  • Synthesises complementary DNA molecule
  • Furhter unwinding of parental duplex at replication fork
  • Extension of the leading strand

Lagging strand synthesised in short bursts:

  • RNA primer, then DNA
  • RNA removed and gap filled by DNA polymerase I
  • Strand joined by ligase
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Fidelity of Replication and Mutations

Fidelity of Replication

E.coli: 1 mistake in 10^10 bases incorporated during replication. This is kept low by:

  • only allowin complementary base pairs to be incorporated
  • proof reading (exonuclease associated with the polymerase)


  • 'mismatched bases', gaps, breaks, T-T dimers
  • DNA is scrutinised continuously, not just during replication.
  • Mistakes are identified
  • DNA polymerase I is brought in to remove and replace
  • DNA ligase to join up
  • Some repairs may fix a mutation (eg mismatched bases)
  • More pressure put on the system; more mistakes are made
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