DNA Replication Mechanism

Important points

• Autocatalytic – DNA itself catalyses the formation of new DNA.
• Semi-conservative – One strand is newly synthesised over an existing parental template strand.
• Semi-discontinuous.
• Template-dependent process.
• Based on complementary base pairing (purine–pyrimidine).

Site of DNA replication

• Occurs in the cytoplasm of prokaryotes, as they lack a membrane-bound nucleus.

Requirements for DNA replication

1) Origin of replication site

• Starting point of DNA replication.
• Prokaryotes have only one ori site in their genome.
• Eukaryotes have multiple ori sites in their genome.

2) Substrates

• Ribonucleoside triphosphates (NTPs) – for RNA primer formation.
• Deoxyribonucleoside triphosphates (dNTPs) – for DNA polymerisation.
• Template DNA – used for complementary base pairing.

3) Enzymes

• Helicase – ATP-dependent unwinding of the DNA helix.
• DNA gyrase – creates nicks to release the supercoiling caused by unwinding.
• Primase – RNA primer formation (DNA-dependent RNA polymerase).
• DNA ligase – joining of DNA fragments.
• DNA polymerase I
  • 5′ → 3′ polymerase activity – for polymerisation.
  • 5′ → 3′ exonuclease activity – for primer removal.
  • 3′ → 5′ exonuclease activity – for proofreading.
• DNA polymerase II
  • 5′ → 3′ polymerase activity – for polymerisation.
  • 5′ → 3′ exonuclease activity – absent.
  • 3′ → 5′ exonuclease activity – for proofreading.
• DNA polymerase III
  • 5′ → 3′ polymerase activity – for polymerisation (main polymerase).
  • 5′ → 3′ exonuclease activity – absent.
  • 3′ → 5′ exonuclease activity – for proofreading.

4) Proteins

• Single-strand binding proteins (SSBP).

5) Ions

• Magnesium ions.

Steps of DNA replication

1) Unwinding of DNA helix

• Starts at the ori site.
• Helicase binds to the ori site and unwinds the double-stranded DNA into single strands.
• Helicase uses ATP to break the H-bonds between the bases, so only short stretches of DNA undergo unwinding, due to the high energy requirement.
• The single strands are stabilised by the binding of SSBP, which prevents them from rejoining.
• Unwinding of DNA results in the formation of the replication fork.

2) Releasing of supercoiling

• Unwinding of DNA at the ori site creates supercoiling in the upstream region.
• Supercoiling is released by DNA gyrase by creating nicks (breaking of phosphodiester bonds) in the DNA and rejoining it.

3) Addition of RNA primers

• The enzyme primase adds a very short fragment of RNA over the template strands to initiate DNA replication.
• RNA primers provide the free 3′-OH group required for the DNA polymerase enzyme to start adding deoxyribonucleotides, based on complementary base pairing.

4) Formation of new strands

• Occurs only in the 5′ → 3′ direction.
• The directional activity of DNA polymerase makes the DNA replication process very complex, as the existing DNA strands have anti-parallel polarity.
• One of the strands is synthesised (over the 3′ → 5′ template strand) continuously and requires only one primer. This strand is called the leading strand, and its synthesis is fast.
• The synthesis of the other strand (over the 5′ → 3′ template) is, however, discontinuous and requires multiple primers. This strand is called the lagging strand, and it is initially synthesised in the form of relatively short fragments of DNA called Okazaki fragments.
• The synthesis of the lagging strand is slow, and it lags behind the leading strand.

5) Removal of RNA primers

• DNA polymerase I removes the RNA primers by its 5′ → 3′ exonuclease activity, and also adds deoxyribonucleotides to replace the RNA primers.
• The Okazaki fragments are now joined by DNA ligase to complete the DNA replication process.