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Taq Polymerase Review

Taq Polymerase

by Daniel Rossie

Figure 1. Jmol Image of Taq Polymerase. 1)


Taq polymerase is an enzyme involved in DNA replication in the organism Thermus aquaticus, whose function is similar to that of DNA polymerase I (pol I). Pol I “functions primarily to fill DNA gaps that arise during DNA repair, recombination, and replication.” 2) DNA Pol I is responsible for replicative DNA synthesis and plays a critical role in DNA repair by proofreading and processing gaps. 3) Taq polymerase functions in much the same way. However, Taq polymerase has been incorporated into a technique called the Polymerase Chain Reaction (PCR) because of its unique properties that enable it to withstand extreme temperatures, while fulfilling the duties that DNA pol I executes. This adaptation to extreme temperatures came out of evolutionary need because the organism Thermus aquaticus is an example of a thermophile and is known for being isolated from a variety of thermal springs in Yellowstone National Park. 4) This species is gram-negative and forms non-sporulating, non-motile rods, which means that they form colonies of rod-shaped, non-moving and non-reproducing groups. Optimal incubation occurs at 70 to 75° C. 5) Since the optimal temperature is considerably high, adaptation of the DNA pol I in Thermus aquaticus must have specialized properties to resist denaturation at these high temperatures. Analysis of this enzyme’s adaptability and function will be the focus of this article, as well as its leading role in PCR.

DNA Polymerase I Family

Figure 2. Superimposition of Taq Pol I (red) and E. coli Pol I (blue). Shows how related Pol I classes have mainly conserved regions across species. 6)

The ability of DNA polymerase I to read DNA and develop a complementary strand is key to replicating DNA. Its major role in DNA replication involves proofreading and excision of mismatched base pairs, including the RNA primers laid down by primase. Prokaryotic Pol I has two functional domains located on the same polypeptide: a 5’-3’ polymerase (located towards the C terminus) and 5’-3’ exonuclease (located towards the N terminus). 7) 8) This enzymatic setup allows for Pol I to carry out its function because at the N-terminus, excision of mismatched base pairs can occur, followed by the polymerase domain making the correct chain needed to replace the excised bases.

DNA polymerase facilitates and catalyzes the same reaction in the polymerase domain, where a base (nucleotide) is added to the 3’ end of the DNA sequence: DNAn + dNTP ↔ DNAn+1 + PPi.9) The three dimensional DNA polymerase proteins have polymerase domains that have “been likened to a right hand with finger, palm and thumb subdomains (Figure 3).”10)

Figure 3. Domain and subdomain organization of Taq DNA polymerase 11)

Structurally, across families of DNA polymerases, the palm domains are relatively similar and all consist of a four- to six- stranded β sheet that has two α helices on one side.12) The structural differences can be seen across families in the finger and thumb domains, except for a commonality in the presence of α helices. Even though there are several structural differences across families of DNAP, it has been shown that these enzymes “play analogous roles.“ 13) The palm domain seems to catalyze a phosphoryl transfer reaction, while the fingers domain involves reactions with the nucleoside triphosphate and the template strand, and the thumb region positions the DNA and is involved in translocation and processing.14) Although the varying structures typically yield a conserved function for DNA polymerases, Taq polymerase has special functioning. While E. coli Pol I contains a 3’-5’ exonuclease domain, species such as Thermus aquaticus do not contain the proper motifs necessary for 3’-5’ exonuclease function.15)16) Let’s now dive into the specific interactions that set apart Taq polymerase.

Taq Polymerase

The central dogma of all science has been portrayed extensively as “structure determines function.” The unique properties of Taq polymerase do not live above this phrase and this enzyme can lend its distinctive functioning to its peculiar structure. “When duplex DNA is bound to homologous T. aquaticus DNA polymerase, which does not contain a functioning exonuclease active site, the 3’ end of the primer strand is found to lie in the polymerase active site adjacent to highly conserved carboxylate residues known to be important for the polymerase reaction.”17) The phosphoryl transfer reaction carried out by all polymerases has been suggested as being catalyzed by a two-metal ion mechanism.18) This mechanism involves interactions of Mg ions with nucleotides (dNTP) in conjunction with three carboxylates.19) “In the homologous 'palm' domains of pol I these two-metal ions (normally magnesium ions) are observed to bind to to the enzyme through two completely conserved carboxylate residues.”20) The metal ions react with the 3' hydroxyl of the primer strand, lowering its pKa. This enables an attack on α-phosphate of the incoming dNTP.21) Overall, the metal ions facilitate stability in the dNTP and in the intermediates throughout the reaction (Figure 4).

Figure 4. Two-metal ion mechanism of DNA polymerase22)

The ability for this reaction to be catalyzed in Taq polymerase is facilitated by the lack of a functioning 3’ to 5’ exonuclease domain which leaves the template strand in the polymerase domain. However, this also leaves this enzyme weak in strand fidelity because of the lack of a proofreading domain found in most DNAPs. This structural anomaly ushers in more DNA into the active site. “The primer terminus of the DNA abuts the fingers domain of Taq pol, and several acidic residues from the palm domain that are responsible for binding the catalytically essential metal ions are located close to the primer. The DNA leads away from the fingers and has several contacts with palm domain. It then encounters the thumb domain, which has extensive contacts with the DNA across its minor groove.”23)

Enzymatic stability in high temperatures is usually attributed to an “abundance of charged and aromatic residues.”24) The carboxylate functional groups and the acidic residues of the palm domain stabilize Taq polymerase at high temperatures because their charges promote intramolecular stabilizing bonds. This gives rise to its functionality in the PCR reaction because its negatively charged residues stabilize the enzyme from denaturing.

“PCR amplification involves two oligonucleotide primers that flank the DNA segment to be amplified and repeated cycles of heat denaturation of the DNA, annealing of the primers to their complementary sequences, and extension of the annealed primers with DNA polymerase.”25) This method for DNA replication is widespread because of its efficiency in duplicating DNA and its practicality in developing gene cloning and expression. The cycling of the thermo cycler brings about a necessity for adaptability in the enzyme to withstand all stages of the cycle. The thermostability of Taq polymerase revolutionized the PCR reaction because of its ability to withstand the denaturation temperature of the thermo cycler and effectively build DNA strands in the elongation cycle.


Taq polymerase has been shown to be an effective DNA polymerase I for the thermophile, Thermus aquaticus. Its practicality stems from its structure being involved as a polymerase, along with its acidic and charged residues giving it its thermostability. Taq polymerase has given PCR its efficiency and ability to analyze DNA more easily. This enzyme is definitely the epitome of the phrase, “Structure determines function.”

2) , 3) , 6) , 7) , 15) Patel, H. et al. (2001). “Prokaryotic DNA Polymerase I: Evolution, Structure and ‘Base Flipping’ Mechanism for Nucleotide Selection”
4) , 5) , 16) Brock, T. et al. (1969). “Thermus aquaticus gen. n. and sp. N., a Non-sporulating Extreme Thermophile”
8) Camps, M. et al. (2003). “When Pol I Goes into High Gear: Processive DNA Synthesis by Pol I in the Cell”
9) , 10) , 11) Pavlov, A. et al. (2004). “Recent developments in the optimization o f thermostable DNA polymerases for efficient applications”
12) , 13) , 23) Brautigam, C. et al. (1998). “Structural and functional insights provided by crystal structures of DNA polymerases and their substrate complexes”
14) , 17) , 18) , 19) , 20) , 21) , 22) Steitz, T. (1999). “DNA Polymerases: Structural Diversity and Common Mechanisms”
24) Bleichert, L. et al. (2011). “Molecular Basis of the Thermostability and Thermophilicity of Laminarinases: X-ray Structure of the Hyperthermostable Laminarinase from Rhodothermus marinus and Molecular Dynamics Simulations”
25) Saiki, R. et al. (1988). “Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase”
chem331/taq_polymerase.txt · Last modified: 2016/06/07 09:53 (external edit)