Question

In DNA polymerases, the 3’ hydroxyl end attacks the phosphorus on the incoming nucleotide triphosphate to add another nucleotide to the growing chain. a) These enzymes require Mg^2+. What function might this cofactor serve in the enzymatic process?    b) How does the mechanism explain the inability of the polymerase to synthesize DNA using nucleotide monophosphates? c) How does the mechanism explain how some DNA polymerases can replace RNA primers and damaged DNA (“nick translation,”) but not seal the backbone (“ligation”)?

Answer 1

a) The catalytic mechanism of DNA polymerase involves two magnesium ions. These ions are coordinated to the phosphate groups of the incoming nucleotide triphosphate and to three Asp residues. The Asp residues are highly conserved and present in all DNA polymerases. One of these ions facilitates the attack of the 3’OH group of the primer on the alpha phosphate of the incoming nucleotide. Another ion is involved in the displacement of the pyrophosphate from the incoming nucleotide.

b) The activity of DNA polymerase requires the presence of free 3' OH group that can serve as a nucleophile during the formation of the phosphodiester bond. This is why it requires a primer with a free 3' OH group.

c) The gaps generated by the removal of primers or damaged DNA have free 3' OH end. The enzyme DNA polymerase extends the free 3' OH by adding dNTPs and thereby fill the nick. The DNA polymerase cannot ligate as the phosphate group present at the 5’ end is not activated. During DNA synthesis, hydrolysis of the pyrophosphate product by the enzyme pyrophosphatase provides part of the free energy required to form the phosphodiester bond. Once the DNA polymerase is removed, nick remains which is sealed by DNA liagse. DNA ligase uses the energy of ATP or NAD+ to activate the phosphate and then catalyzes the formation of a phosphodiester bond.