(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.