DNA Replication

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 DNA
Replication
DNA
Reverse Transcription
Transcription
Translation
RNA RNA protein
Replication

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Genetic Information Must Be Accurately Copied Every
Time a Cell Divides

• Replication has to be extremely accurate:

• 1 error/million bp leads to 6400 mistakes

every time a cell divides, which would be
catastrophic.

• Replication also takes place at high speed:

• E. coli replicates its DNA at a rate of 1000

nucleotides/second.
Replication Models:

• Conservative replication model

• Dispersive replication model

• Semiconservative replication
Meselson and Stahl’s Experiment

• Two isotopes of nitrogen:

• 14N common form; 15N rare heavy form

• E. coli were grown in a 15N media first, then

transferred to 14N media.

• Cultured E. coli were subjected to equilibrium

density gradient centrifugation.
Meselson and Stahl (1958)

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Modes of Replication

• Replicons: units of replication

• Replication origin

• Theta replication: circular DNA, E. coli; single

origin of replication forming a replication fork,
usually a bidirectional replication

• Rolling-circle replication: virus, F factor of

E. coli; single origin of replication
Linear Eukaryotic Replication

• Eukaryotic cells; thousands of origins; a typical

replicon: 200,000 ~ 300,000 bp in length
 Uni or bidirectional
Replication forks move in one or opposite directions
◼
Linear Eukaryotic Replication

• Requirements of replication:

• A template strand

• Raw material: nucleotides

• Enzymes and other proteins

Linear Eukaryotic Replication

• Direction of replication:

• DNA polymerase add nucleotides only to the

3′ end of a growing strand.

• The replication can only go 5′→3′.

Linear Eukaryotic Replication
• Direction of replication:

• Leading strand: undergoes continuous

replication

• Lagging strand: undergoes discontinuous

replication

• Okazaki fragment: the discontinuously

synthesized short DNA fragments
forming the lagging strand
 Semi-discontinuous replication
Anti parallel strands replicated simultaneously
❑ Leading strand synthesis continuously in 5’– 3’
❑ Lagging strand synthesis in fragments in 5’-3’
Core proteins at the replication fork

Topoisomerases - Prevents torsion by DNA breaks

Helicases - separates 2 strands
Primase - RNA primer synthesis
Single strand - prevent reannealing
binding proteins of single strands
DNA polymerase - synthesis of new strand
Tethering protein - stabilises polymerase
DNA ligase - seals nick via phosphodiester
linkage
The mechanism of DNA replication
Arthur Kornberg, a Nobel prize winner and
other biochemists deduced steps of
replication
– Initiation
• Proteins bind to DNA and open up double helix
• Prepare DNA for complementary base pairing
– Elongation
• Proteins connect the correct sequences of
nucleotides into a continuous new strand of DNA
– Termination
• Proteins release the replication complex
Supercoiled DNA relaxed by gyrase & unwound by helicase + proteins:

5’ SSB Proteins
Okazaki Fragments
ATP
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Polymerase III 2 Helicase

Lagging strand 3 +
Initiator Proteins

3’
primase base pairs

Polymerase III 5’
RNA primer replaced by polymerase I

& gap is sealed by ligase

Leading strand
RNA Primer

3’ 23
DNA ligase seals the gaps between Okazaki fragments with a
phosphodiester bond

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Model of DNA replication

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Model of DNA replication

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See you in the next lecture
With my best wishes

THANK YOU
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