Question

1. Write chemical reactions for both DNA polymerization activity and 3'-->5' exonuclease activity of a DNA polymerase. Are the 2 reactions reversible to each other? Explain why 3'-->5' exonuclease activity is essential for a high fidelity DNA polymerase.

Answer 1

1. DNA polymerization is basically the process of extending the DNA by adding the nucleotides which are basically the building blocks or monomer of DNA. The polymerization is a nucleophilic attack by a 3'-OH group of the primer on the innermost P atom of the deoxyribonucleoside triphosphate (dNTP).

A phosphodiester bond forms with the concomitant release of pyrophosphate. The catalytic Mg+2 ion interacts with the 3'-OH resulting in reduced association between the O and H. This leaves a nucleophilic 3-O- which attack the innermost P atom of the dNTP as described above. Metal ion also interacts with the triphosphates of the incoming dNTP to neutralize their negative charge.

So the net reaction is: [dNMP]n+dNTP-----> [dNMP]n+1+ PPi (Pyrophosphate).

Now coming to 3' to 5' exonuclease activity, it serves role in mismatch repairing or fidelity.Because of this activity the DNA polymerase remove nucleotides that were added in error by hydrolyzing the phosphodiester bonds it has just made (if the wrong base was put in). Net reaction:

chain (n+1 units long) + H2O ↔ chain (n units long) + dNMP; X= Nitrogenous base.

No, these two reactions are not reversible to each other because the exonuclease activity basically catalyzes the hydrolysis of phosphodiester bond and NMP releases but in case of polymerization what we have on the left side is dNTP. So thats how we can say that these two reactions are not reversible to each other.

Fidelity in this process refers to the ability of the polymerase to avoid or to correct errors in the newly synthesized DNA strand. The 3' to 5' exonuclease activity is essential for high fidelity because with the help of this activity the DNA polymerase is going to identify the mismatched or mispaired nucleotides and remove those nucleotides by hydrolyzing the phosphodiester bonds and fill the gap using its DNA polymerization activity (5' to 3'). So because of this we can say that the exonuclease activity is essential for a high fidelity DNA polymerase.