DNA dependent DNA polymerase (DNAP of prokaryotes)
DNA polymerases are the enzymes involved in DNA synthesis from deoxyribonucleotides.
DNA polymerase adds nucleotides to the 3′- end of a DNA strand, one nucleotide at a time.
Chemical reaction catalysed:
What do you mean by processivity of DNA polymerase enzyme?
- The processivity of a DNA polymerase is the number of nucleotides added by the polymerase each time it binds to the template.
- The processivity of DNA polymerases vary with the type of polymerase and can range from few nucleotides to as many as 50,000.
- The processivity of DNA polymerases is increased by their association with the sliding clamps.
Structure of DNA polymerase from E. coli
The shape of the DNA polymerase resembles a right hand with thumb, finger, and palm domains.
1) The palm domain: The active site of polymerase resides in the palm domain. DNA is bound to the palm when the enzyme is active. Composed of β-sheet and binds two divalent metal ions like Mg2+ or Zn2+
Function: Catalyzing the transfer of phosphoryl groups in the phosphoryl transfer reaction.
2) The finger domain: The nucleotide recognition and binding with the template base.
3) The thumb domain: Plays a potential role in the processivity, translocation, and positioning of the DNA. Important role in binding of substrate DNA with polymerase enzyme.
Common features of all class of prokaryotic DNA polymerases
- Synthesize any sequence specified by the template strand.
- Catalyze new strand synthesis in the 5’ to 3’ direction by adding nucleotides to a 3’-OH group.
- Use dNTPs for synthesizing new DNA.
- Require an RNA primer to initiate DNA synthesis.
- Catalyze the formation of a phosphodiester bond by joining the 5’ phosphate group of the incoming nucleotide to the 3’-OH group of the preceding nucleotide on the growing strand. In this process two phosphates are cleaved off
- New strands produced by DNPS are complementary and antiparallel to the template strands.
- They are associated with a number of other proteins.
Proof reading activity of DNA polymerase
- DNA polymerase has an error-correcting activity. This activity is known as proofreading.
- DNA polymerase is so accurate in its action. It makes only about one error in every 10^7 nucleotide pairs it copies.
- If an incorrect nucleotide is added to a growing strand, the DNA polymerase will remove it from the strand and replace it with the correct nucleotide before continuing DNA synthesis.
- Before the enzyme adds a nucleotide to a growing DNA chain, it checks whether the previous nucleotide added is correctly base-paired to the template strand. If it is correct,the polymerase adds the next nucleotide.
- If base pairing is not correct, the polymerase removes the mis paired nucleotide by cutting the phosphodiester bond it has just made. As a result mis paired nucleotide is released. DNAP tries again adding correct base.
- Thus, DNA polymerase possesses both a 5’ to 3’ polymerization activity and a 3’ to 5’ exonuclease activity. These activities are carried out by different domains within the polymerase molecule.
(Refer my lecture notes on types of prokaryotic DNA polymerase and DNA Polymerase III holoenzyme for more detailed explanation)