Biowiz

DNA REPLICATION

Updated: Feb 1

(in prokaryotes)

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.

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