DNA Polymerase

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Introduction

DNA polymerase is a ubiquitous enzyme responsible for synthesizing complementary DNA strands using DNA in living cells as templates. Many enzymes have been identified from each organism, and numerous studies have been conducted to investigate the shared functions of these enzymes. In addition to its role in maintaining genome integrity during replication and repair, DNA polymerases can also play important roles in DNA manipulation in vitro, such as DNA cloning, sequencing, labeling, mutagenesis, and more. The ability of DNA polymerases to synthesize deoxyribonucleotide chains is conserved. However, its processivity, fidelity (synthesis accuracy) and substrate nucleotide selectivity vary significantly among the enzymes. Utilizing the different and unique properties of each DNA polymerase can aid the development of unique reagents, so the search for new DNA polymerases has been one of the major focuses of this research field. In recent years, a variety of powerful DNA polymerases have been successfully developed using protein engineering techniques for a variety of specific purposes in DNA manipulation. Thermostable DNA polymerases are particularly important for PCR-related techniques in molecular biology.

Figure 1. Distribution of DNA polymerases in the three domains of life (Ishino, S.; Ishino, Y. 2014)

In the Beginning: Taq Polymerase

The most representative thermostable DNA polymerase is DNA polymerase I from Thermus aquaticus, namely Taq polymerase. Taq polymerase was originally identified from T. aquaticus isolated in Yellowstone National Park, Montana, USA, when it was published by Chien et al., however no one could have foreseen that this enzyme would become so famous. The PCR (polymerase chain reaction) technique using the Klenow fragment of DNA polymerase I from Escherichia coli was reported in 1985. Heat-stable DNA polymerases that do not inactivate during the step of denaturing double-stranded DNA to single-stranded DNA have played a crucial role in transforming the PCR method into a wide range of practical technique. Subsequently, a simple and reliable PCR method using Taq polymerase was invented. Due to the superior thermal stability of Taq polymerase, after preparing the reaction mixture containing DNA polymerase, the reaction tube could remain in the incubator, and only need to change the temperature during the experiment.

PCR kits and instruments capable of rapidly changing reaction temperatures opened up the PCR market. Taq polymerase was purified from T.aquaticus cells, and then the pol gene was cloned from the T.aquaticus genome and successfully expressed in E. coli cells. Commercially, the native Taq polymerase was replaced by a recombinant Taq polymerase called AmpliTaq DNA polymerase. The amount of recombinant Taq polymerase produced in E. coli cells was very low due to the low expression of the T. aquaticus gene, with high GC content (70%). It was later optimized to improve the Taq polymerase production more than 10-fold. A large amount of PCR data using Taq polymerase has been accumulated, providing a valuable resource for the development of new products for PCR modifications.

Protein Engineering of Thermostable DNA Polymerases

Taq polymerase is a family A enzyme that can be used for practical dideoxy sequencing. However, the output of Taq polymerase sequencing data was less than ideal compared to the results from T7 DNA polymerase (Sequenase). Using an ingenious protein engineering strategy, the scientists arrived at a mutant Taq polymerase that was more suitable for dideoxy sequencing than wild-type Taq polymerase.

E. coliPolI and Taq polymerases discriminate deoxy- and dideoxynucleotides as substrates for incorporation into DNA strand. T7 DNA polymerase equally incorporates deoxynucleotides and dideoxynucleotides, making it easier to obtain very clear signals. A T7 DNA polymerase lacking 3'-5' exonuclease activity was developed as a commercial product, named Sequenase.

The "domain tagging" strategy can successfully produce improved DNA polymerases. For example, new DNA polymerases are generated by linking the helix-hairpin-helix (HhH) domain of Methanopyrus kandleri topoisomerase V to the catalytic domains of Taq polymerase and Pfu polymerase. HhH acts on sequence-nonspecific DNA binding. These hybrid enzymes have improved properties, such as enhanced thermostability, and strong resistance to salts and inhibitors such as blood, phenol, and DNA intercalating dyes. Another successful example of a tagging strategy is the creation of the commercial product "Phusion DNA Polymerase". This is a fusion protein of Pfu DNA polymerase and the DNA binding protein Sso7d, from S. solfataricus. Compared to Pfu DNA polymerase, this enzyme has relatively high extension rates while maintaining high fidelity. This enzyme shows very high processivity and accurate PCR performance, so it is widely used.

Figure 2. Schematic diagrams of processive PCR using a family B DNA polymerase (Ishino, S.; Ishino, Y. 2014)