IMPORTANT POINTS

• Autocatalytic- DNA itself catalyzes the formation of new DNA.
• Semi-conservative- One strand is newly synthesized over an existing parental template strand.
• Semi-discontinuous.
• Template dependent process.
• Based on complimentary 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 polymerization.
  • Template DNA- Used for complimentary base pairing.

3) ENZYMES

• • Helicase- ATP dependent unwinding of DNA helix.
  • DNA gyrase- Creates nicks to release 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 polymerization.
    • 5′-> 3′ exonuclease activity- for primer removal.
    • 3′-> 5′ exonuclease activity- for proof reading.
  • DNA polymerase II
    • 5′-> 3′ polymerase activity- for polymerization.
    • 5′-> 3′ exonuclease activity- absent
    • 3′-> 5′ exonuclease activity- for proof reading.
  • DNA polymerase III
    • 5′-> 3′ polymerase activity- for polymerization. (main polymerase)
    • 5′-> 3′ exonuclease activity- absent
    • 3′-> 5′ exonuclease activity- for proof reading.

4) PROTEINS

• • Single Strand Binding Proteins (SSBP)

5) IONS

• • Magnesium ions.

STEPS OF DNA REPLICATION

1) Unwinding of DNA helix

• • Starts at Ori site.
  • Helicase binds to the Ori site and unwinds the double stranded DNA into single strands.
  • Helicase uses ATP to break H-bonds between the bases, so only short stretches of DNA undergoes unwinding due to high energy requirements.
  • Single stands are stabilized by binding of SSBP to prevent it from rejoining.
  • Unwinding of DNA results in formation of Replication fork.

2) Releasing of supercoiling

• • Unwinding of DNA at 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

• • Enzyme Primase adds a very short fragment of RNA over the template strands to initiate DNA replication.
  • RNA primers provides the free -OH group required for DNA polymerase enzyme to start adding deoxyribonucleotides based on complimentary base pairing.

4) Formation of new strands

• • Occurs in only 5′-> 3′ direction.
  • Directional activity of DNA polymerase makes the DNA replication process very complex as the existing DNA strands have anti-parallel polarity.
  • One of the strand is synthesized (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 other strand (over the 5′-> 3′ template) is however discontinuous and requires multiple primers. This strand is called Lagging Strand and is initially synthesized in the form of relatively short fragments of DNA called Okazaki fragments.
  • The synthesis of Lagging strand is slow and it lags behind the Leading strand.

5) Removal of RNA primers

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