DNA polymerase is a ubiquitous enzyme that synthesizes complementary DNA strands based on template DNA in living cells. Various enzymes with this function have been identified across organisms, and their shared functions have been extensively investigated. Beyond their essential role in maintaining genome integrity during replication and repair, DNA polymerases have found extensive use in in vitro DNA manipulation, including cloning, sequencing, labeling, mutagenesis, and more. While the fundamental ability to synthesize a deoxyribonucleotide chain is conserved among DNA polymerases, specific properties like processivity, fidelity, and substrate nucleotide selectivity vary among enzymes.
Related Products
Fig. 1 DNA polymerase.
The Emergence of Taq Polymerase
Taq polymerase, initially purified from T. aquaticus cells, played a pivotal role in the revolutionary polymerase chain reaction (PCR) technology. The heat stability of Taq polymerase enabled PCR reactions to proceed without enzyme inactivation, facilitating the development of quick temperature-changing instruments. In the early PCR era, Taq polymerase was cloned from the T. aquaticus genome and expressed in Escherichia coli, leading to the commercial product AmpliTaq DNA polymerase. However, its production was limited due to low expression of the T. aquaticus gene. Optimization of codons around the N-terminal region significantly increased Taq polymerase production. Despite the emergence of other DNA polymerases, Taq polymerase has remained the standard for PCR.
Thermostable DNA Polymerases from Thermophiles
Thermophiles, organisms thriving at high temperatures, offer a rich source of thermostable DNA polymerases. These enzymes play a pivotal role in various applications, and their heat stability is directly correlated with the organism's growth temperature. Hyperthermophiles, extreme thermophiles growing optimally above 80°C, particularly those from Archaea, have demonstrated superior heat resistance compared to normal thermophiles. The emergence of DNA polymerases like Pfu polymerase from Pyrococcus furiosus showcased enhanced stability compared to Taq polymerase, contributing to advancements in both industrial applications and fundamental molecular biology research.
DNA Polymerases from Hyperthermophiles
The search for thermostable DNA polymerases has expanded beyond the boundaries of individual organisms, and the family A enzyme, Thermotoga maritima DNA polymerase, was among the first commercial products from hyperthermophilic bacteria. However, despite its proofreading activity, it did not achieve commercial success. Research on DNA polymerases from hyperthermophilic archaea, such as Pfu polymerase, revealed sequences similar to eukaryotic replicative DNA polymerases. This family B enzyme exhibited higher accuracy in DNA synthesis compared to Taq polymerase, providing valuable alternatives for specific applications.
Development of Long and Accurate PCR (LA-PCR)
The demand for accurate PCR amplification of long DNA regions led to the development of long and accurate PCR (LA-PCR). By combining an N-terminal deletion mutant of Taq polymerase (Klentaq1) and an archaeal family B DNA polymerase with 3'-5' exonuclease activity, researchers successfully amplified a~35 kb DNA fragment from λ phage genomic DNA in 1994. LA-PCR technology rapidly gained popularity, with commercial products emerging to facilitate the amplification of longer DNA fragments.
Fast and Highly Accurate PCR by an Archaeal Family B DNA Polymerase
The identification of a family B DNA polymerase from the hyperthermophilic archaeon Thermococcus kodakarensis, later commercialized as KOD DNA polymerase (KOD Pol), introduced an enzyme with high extension rates and fidelity. The unique structural features of KOD Pol, with numerous basic residues around the active site, contributed to its efficient extension ability. The success of KOD Pol spurred the development of related products, including hot start kits and LA-PCR kits.
Basic Research on Archaeal DNA Polymerases
While the industrial applications of DNA polymerases are vast, basic research on these enzymes has provided valuable insights into the molecular biology of hyperthermophilic archaea. Studies on P. furiosus unveiled a novel DNA polymerase, PolD, consisting of two proteins (DP1 and DP2), marking the first report of a eukaryotic-like initiator protein for DNA replication in archaea. The subsequent identification of PolD in other archaea solidified its status as a distinct family (family D) within DNA polymerases.
The evolution of DNA polymerases, particularly thermostable enzymes, has significantly contributed to molecular biology and biotechnology. From the groundbreaking discovery of Taq polymerase to the exploration of hyperthermophilic archaeal DNA polymerases, researchers have harnessed the unique properties of these enzymes for various applications. The continuous development of specialized DNA polymerases, along with protein engineering techniques, promises continued advancements in the field of DNA manipulation. As our understanding of the structure-function relationships of DNA polymerases deepens, the design and optimization of enzymes for specific tasks will likely continue to expand, opening new possibilities for genetic research and biotechnological applications.
Reference
- Ishino S., Ishino Y. DNA polymerases as useful reagents for biotechnology-the history of developmental research in the field. Frontiers in Microbiology. 2014, 5: 465.
.
