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This picture on the right shows a small template DNA molecule bound to the active site of Taq polymerase |
General Mechanism
The mechanism for most DNA polymerases are very similar. Like all DNA polymerases, Taq polymerase too reads in 3'->5' direction, and synthesizes DNA in the 5'->3' direction, adding one nucleotide at a time to the 3' end of the growin chain. The incoming dNTP binds to its complementary residue on the template strand. This is followed by a nucelophilic attack on the incoming dNTP, leading to addition of a nuceloside monophosphate and displacement of pyrophosphate. The pyrophosphate is hydrolyzed by the enzyme pyrophosphatase, and this step ensures that the reaction is irreversible.
Image 6: Mechanism of elongation of the DNA chain
Since the two strands of DNA run anti-parallel, the synthesis of one strand (the leading strand) occurs in the same direction as the fork movement. while the synthesis of the other strand (the lagging strand) occurs in the opposite direction, in a discontinuous fashion.
Unique to Taq Polymerase:
It is proposed that like some other DNA polymerases, Taq polymerases works using a two divalent metal-ion mechanism (mostly magnesium ions.) According to this mechanism, one of the metal ions interacts with the hydroxyl group of the primer strand. deprotonating it by reducing its pKa. This makes it a better nucleophile, making it easier to attack the incoming dNTP (similar to the chymotrypsin mechanism.) The second metal ion stabilizes the negative charge in the transition state that occurs in this reaction. In addition, it also facilitates the leaving of the phosphate groups from the dNTP.
Taq polymerase is known to have a relatively high processivity rate, meaning it replicates DNA rather quickly. However, this comes at the price of a reduced fidelity when compared to other polymerases; the DNA copies made by Taq polymerase are not the most accurate copies of the template.
The mechanism for most DNA polymerases are very similar. Like all DNA polymerases, Taq polymerase too reads in 3'->5' direction, and synthesizes DNA in the 5'->3' direction, adding one nucleotide at a time to the 3' end of the growin chain. The incoming dNTP binds to its complementary residue on the template strand. This is followed by a nucelophilic attack on the incoming dNTP, leading to addition of a nuceloside monophosphate and displacement of pyrophosphate. The pyrophosphate is hydrolyzed by the enzyme pyrophosphatase, and this step ensures that the reaction is irreversible.
Image 6: Mechanism of elongation of the DNA chain
Since the two strands of DNA run anti-parallel, the synthesis of one strand (the leading strand) occurs in the same direction as the fork movement. while the synthesis of the other strand (the lagging strand) occurs in the opposite direction, in a discontinuous fashion.
Unique to Taq Polymerase:
It is proposed that like some other DNA polymerases, Taq polymerases works using a two divalent metal-ion mechanism (mostly magnesium ions.) According to this mechanism, one of the metal ions interacts with the hydroxyl group of the primer strand. deprotonating it by reducing its pKa. This makes it a better nucleophile, making it easier to attack the incoming dNTP (similar to the chymotrypsin mechanism.) The second metal ion stabilizes the negative charge in the transition state that occurs in this reaction. In addition, it also facilitates the leaving of the phosphate groups from the dNTP.
Taq polymerase is known to have a relatively high processivity rate, meaning it replicates DNA rather quickly. However, this comes at the price of a reduced fidelity when compared to other polymerases; the DNA copies made by Taq polymerase are not the most accurate copies of the template.