Types of DNA polymerases in prokaryotes

Types of DNA polymerases in prokaryotes

Prokaryotes consist of five DNA polymerases of which only Pol III is involved in DNA replication.

The first polymerase to be identified was Pol I from E. coli. The enzyme was isolated by Arthur Kornberg.

DNA polymerase I

  • Encoded by the polA structural gene. Molecular weight is 103,000 Dalton
  • Pol I is the most abundant polymerase.

Polymerization rate: Pol I adds ~15-20 nucleotides per second.

Processivity: poor processivity (3 -200 nucleotides are added before it dissociates from the DNA template)

Key Features:

  • 3’ to 5’ Exonuclease activity
  • 5’ to 3’ Exonuclease activity
  • 5’ to 3’ Polymerase activity

Functions:  

Primer removal:

  • 5’ to 3’ exonuclease removes the RNA primers at the 5’ ends of newly synthesized DNA while its DNA polymerase activity fills in the resulting gaps.
  • DNA polymerase I degrade both single and double stranded DNA in the 5’ to 3’ direction using the 5’ to 3’ exonuclease activity, yielding 5’-mononucleotides.
  • The 5’ to 3’ exonuclease activity is specific for double stranded DNA, yielding 5’-mononucleotides and oligonucleotides.
  • DNA polymerase I can also excise mismatched regions in DNA.

Proof reading: If DNA polymerase III makes a mistake during DNA synthesis, it will remove the resulting unpaired base at 3′ end at the growing strand. After that DNA synthesis continues.

Polymerization:

  • Polymerase I starts adding nucleotides at  the origin of replication (ori). The Pol III holoenzyme is assembled approximately 400 bp downstream from the origin and takes over replication at a highly processive manner.
  • Fill gaps in DNA that arise during DNA replication, repair, and recombination.

Proteolytic cleavage of DNA polymerase I

  • Polymerase I contains three separate active sites on a single polypeptide chain.
  • The 5’ to 3’ exonuclease activity of Pol I is independent of its 3’ to 5’ exonuclease and its polymerase activities.
  • The 5’ to 3’ exonuclease domain of the pol I can be removed by mild proteolytic treatment.

Protease used: Trypsin or subtilisin (cleavage site: peptide bond between 323rd and 324 th aminoacid residues)

Cleavage products: Smaller fragment (35KD) + Larger fragment (68 KD)

Figure 1: Diagrammatic representation of Proteolytic cleavage of DNA polymerase I
  • The larger C terminal fragment is known as Klenow fragment.
  • Klenow fragment retains its 3’ to 5’ exonuclease activity and 5’ to 3’ polymerase activity. But it lacks 5’ to 3’ exonuclease activity.
  • Aminoacid residues in Klenow fragment: 324th aminoacid to 928th aminoacid

Smaller N terminal fragment has only 5’ to 3’ exonuclease activity

Aminoacid residues in smaller fragment: 1st aminoacid to 323 rd aminoacid

DNA polymerase II

  • Encoded by structural gene polB.
  • Molecular weight: 88000 Dalton
  • Polymerization rate: 40 nucleotides/ sec
  • Processivity: ~ 1,500 nucleotides are added before it dissociate from template

Key features:

  • 3’ to 5’ exonuclease activity: Proof reading function
  • 5’ to 3’ polymerase activity

Functions

  • Capable of synthesizing DNA on a damaged template strand (replication restart in UV-irradiated E.coli)
  • Carry out DNA synthesis when pol I and pol III are not functional.
  • Participate in DNA repair (repair of DNA damaged by UV irradiation)

DNA polymerase III

  • Encoded by structural gene polC (dnaE)
  • Polymerization rate: 1000 nucleotides/ s
  • Processivity: ~ 500,000 nucleotides are added before it dissociates from template.

Key features:

  • 3’ to 5’ exonuclease activity: Proof reading function
  • 5’ to 3’ polymerase activity : Responsible for elongation process

(Refer my lecture notes on DNA polymerase III holoenzyme for more details)

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