The thermostable DNA-dependent DNA polymerase I, Taq DNA polymerase (Taq pol), is the most extensively used DNA polymerase for amplifying genetic material by the polymerase chain reaction (PCR) (1) and for sequencing DNA. Taq pol is obtained from Thermus aquaticus, an archeabacterium that thrives at elevated temperatures in deep thermal vents, and the enzyme is highly resistant to denaturation at elevated temperatures. Taq DNA polymerase is closely related to DNA polymerase I of Escherichia coli in both its primary and three-dimensional, tertiary structures. Both are single subunit enzymes with C-terminal polymerase and N-terminal 5′-3′ exonuclease domains. However, Taq pol lacks the 3′-5′ exonuclease (proofreading) function found in E. coli pol I and therefore cannot excise polymerization errors incurred during synthesis (2).
1. Properties
Taq pol has been cloned and overexpressed in E. coli The purified full-length protein is composed of a single subunit of 94 kDa with two distinct activities: 5′-3′ polymerase and 5′-3′ exonuclease. Taq DNA polymerase is highly thermostable, loses only 10% of its activity after 30 min incubation at 72 °C (which is the normal growth temperature for Thermus aquaticus), and 50% activity after 30 minute incubation at 95 °C (M. Suzuki and L.A. Loeb, unpublished results). Like DNA pol I of E. coli, Taq pol is susceptible to proteolytic cleavage and yields a small N-terminal fragment that has 5′-3′ exonuclease activity and a large C-terminal fragment that has polymerase activity. The large C-terminal fragment is analogous to the Klenow fragment of DNA pol I and is also called the KlenTaq or Stoffel fragment. It is thermostable, loses only 10% activity after 30 min incubation at 95 °C, and therefore is frequently used in polymerase chain reactions (PCR) and various sequencing protocols that involve prolonged incubations at elevated temperatures.
Taq pol conducts DNA-templated DNA synthesis with moderate accuracy, on average misincorporating only 1 in 9000 nucleotides (2). The predicted fidelity during PCR is one error per 400 bases after 25 cycles. The fidelity during PCR is enhanced twofold by using the truncated KlenTaq fragment instead of full-length Taq pol (3) and up to 10-fold by using other thermostable polymerases that have a 3-5′ exonuclease (proofreading) function. Specific methods that enhance the fidelity of Taq pol include incubation at low pH (5 to 6) or with reduced concentrations of MgC^.
However, both of these methods decrease Taq pol activity and are generally incompatible with PCR, especially for amplifying long fragments.
The two components of fidelity include (1) nucleotide misinsertion and (2) extension of the misinserted base. Taq pol extends mispairs at a very low efficiency: 10-3 for a T-G mispair to 10-6 for A-A mispair (4). Because of its relative specificity to extend only properly matched primers, Taq pol is frequently used to detect specific in vivo mutations. 2. Structure
The X-ray crystallographic structures of all polymerases determined to date, including Taq DNA polymerase, resemble in overall morphology a cupped human right hand, complete with subdomains corresponding to the fingers (which bind the single stranded-template), palm (which binds the incoming deoxynucleoside triphosphate, DNTP) and thumb (which binds double-stranded DNA) (5, 6) (Fig. 1). The polymerase subdomains of Taq pol and the Klenow fragment are highly homologous and have 51% amino acid identity. In addition, the overall folding of the two polymerase domains in the three-dimensional structures are virtually identical and have an average (root mean square) deviation in alpha carbons of approximately 1.4 A. The overall folding and location of the 3′-5′ exonuclease domain, which is 30 A away from the polymerase active site, is also similar in both enzymes. Taq pol, however, lacks four loops found in the Klenow fragment on one side of the 3′-5′ exo site (5). In addition, four key amino acid residues believed to bind metal cofactors involved in the exonuclease reaction in the Klenow fragment (Asp424, Asp501, Asp355, and Glu357) are replaced by nonacidic residues in Taq pol (Leu356, Arg405, Gly308, Val310). These changes may explain why the 3′-5′ nuclease domain of Taq pol is inactive. A direct comparison of the Klenow and Taq pol structures also shows that Taq contains a more hydrophobic core and more favorable electrostatic interactions. Both of these properties may contribute to the greater thermostability of Taq pol (6).
Figure 1. Ribbon diagram of the Taq DNA pol structure in the absence and presence of double-stranded DNA. This poly cupped human right hand. Residues of helix O located in the finger subdomain probably interact with the extended single I interact with the duplex portion of the DNA. The structure contains an inactive, vestigial 3′-5′ nuclease proofreading dc catalytic site, and a functioning 5′-nuclease site located 70 A from the active polymerase site.
Interestingly, the location of the 5′-3′ exonuclease active site, which probably functions in nick translation during DNA repair and during removal of Okazaki Fragments, is 70 A from the polymerase active site. How polymerase and 5′-3′ exonuclease sites so far apart work in concert to leave a "repaired" double-stranded DNA with only a nick remains a mystery. The structure of Taq pol bound to blunt-end DNA has been described (7) (Fig. 1). Similar to the complexes with DNA of pol b-DNA and HIV reverse transcriptase, the DNA in the Taq pol active site adopts a structure that is a hybrid between A and B forms. As a consequence, the minor groove, which interacts with amino acid residues in the polymerase active site, is especially wider than in B-form DNA. It is thought that protein side-chains may form hydrogen bonds with the O2 atom of the pyrimidine ring and with the N3 atoms of purines (7). Because the positions of these two atoms are unchanged in A:T and G:C base pairs, these putative hydrogen-bond interactions between protein side chains and the last base pair may insure proper base pairing before nucleotide incorporation and thus enhance the fidelity of the polymerase.
3. Uses
Taq pol is used extensively in polymerase chain reactions to amplify genetic material. PCR involves incubation with the gene that is to be amplified in the presence of a polymerase, PCR primers that flank the gene of interest, all four dNTP, and magnesium . Briefly, each cycle of PCR involves three steps: (1) incubation at elevated temperature to separate the double-stranded DNA (typically 95°C); (2) incubation at lower temperature to allow primer/template annealing, and (3) incubation at a temperature for polymerization (1). Before the discovery of Taq pol, PCR involved manually adding a small amount of polymerase to the incubation mixture before each polymerization step because the polymerization was inactivated during incubation at elevated temperatures. The thermostability of Taq allows heat denaturation of DNA after each cycle without enzyme inactivation, thus allowing automation of PCR. In general, current PCR technology using full-length Taq pol allows amplification from one molecule to more than 105 copies of a target sequence, which can be as large as 5 kilobases. Even larger sequences are amplified by combining a truncated KlenTaq fragment with low levels of a thermostable polymerase that contains 3′-5′ proofreading activity (eg, Vent DNA polymerase or Pfu polymerase) (8).
PCR is used widely in research and clinical laboratories for diverse procedures including amplifying genes for cloning, diagnosis of diseases, and detecting levels of viruses. Taq DNA polymerase, which possesses weak RNA-templated DNA polymerase activity, is also used to synthesize double-stranded DNA from messenger RNA templates (9). However, this process, termed RT-PCR, is generally more efficient if a reverse transcriptase is used during the first cycle to generate single-stranded DNA and Taq pol is used in subsequent amplification cycles (10). Alternatively, a polymerase from the thermophilic bacteria Thermus thermophilus (TTH POL), which contains efficient RNA-templated and DNA-templated DNA polymerase activities in the presence of Mn , can be used for RT-PCR (11). Because of its relative specificity to extend only properly matched primers, Taq pol I is used to detect specific mutations through PCR analysis, simply by choosing primers with a 3′ -terminus that is complementary to the mutant. Efficient amplification of the DNA in this procedure suggests the presence of a specific mutation. This protocol has been used successfully to detect mutations in the ras oncogene and common mutations resulting in inherited disorders.
3.1. Taq Pol in DNA Sequencing
Until recently, enzymatic DNA sequencing using Taq pol was marred because dideoxynucleotides (DDNTP) are not efficiently incorporated by Taq pol (its usage of ddNTP is 1000 times less efficient than that of dNTP in the presence of Mg ; Ref. 12). However, it has been recently shown that the substitution Phe667Tyr of Taq pol increases incorporation of ddNTP relative to dNTP 250 by 8000-fold (13). This mutated Taq (Thermo sequanase) enables cycle sequencing, thus producing accurate and analyzable sequences using either radioactive or fluorescent sequencing technologies
3.2. Taq Pol in T/A Cloning
Taq pol shares a characteristic common to all polymerases that lack 3′-5′ exonuclease proofreading activity in that it incorporates nontemplated nucleotides (usually adenine) onto blunt-end DNA (15). This property is frequently used in molecular biology to ligate fragments of DNA in T/A cloning protocols. Briefly, PCR amplification of the sequence of interest results in a significant proportion of products containing a nontemplated adenine incorporated onto both 3′ ends. Then this PCR product is incubated in the presence of a ligase with a linear vector that contains 3′ thymine residues. The completed reaction results in a circular DNA that contains a cloned insert.
