They have also been crucial to the development of DNA sequencing and PCR, upon which much of modern biotechnology is built. Today polymerases are the core tools for DNA labelling, sequencing and amplification. DNA polymerases are also intrinsic components for the development of molecular diagnostics for personalised medicine.
They are at the forefront, for example, of techniques to detect genomic alterations that can cause diseases like cancer or cause patients to experience adverse reactions to drugs. The history of DNA polymerase is rooted in the work of Arthur Kornberg who in discovered that an enzyme he extracted from potatoes nucleotide pyrophosphatase could synthesise Nicotinamide adenine dinucleotide NAD , a coenzyme found in all living cells.
Soon after he discovered more enzymes that could synthesise other coenzymes. Having found an enzyme that incorporated a nucleotide into a coenzyme, in Kornberg began to investigate whether there were other enzymes that could assemble the many nucleotides that make up the chains of nucleic acids, particularly RNA. The difficulty he faced was that at this stage scientists did not know what the actual building blocks of nucleic acids were.
By Kornberg's research and that of others had shown that the likely nucleotide building blocks of nucleic acids were synthesised and activated in cells.
Based on this he and his post-doctoral fellow Uri Littauer, launched some experiments to test the power of extracts from Escherichia coli E coli to synthesise RNA. This they performed with a radiolabelled coenzyme called adenosine triphosphate ATP. Their work, however, was to take a new direction when, in , Mariane Grunberg-Manago, a postdoctoral fellow in Severo Ochaoa's laboratory, announced the identification of a new enzyme, polynucleotide phosphorylase. This she had stumbled upon while conducting research to understand aerobic phosphorylation in extracts of Azotobacter vinelandii, a soil dwelling organism.
Importantly, the enzyme was shown to be capable of synthesising RNA in a test tube from simple nucleotides. This she had done with the use of the coenzyme Adenosine diphosphate ADP. This had been a difficult and demanding task, hindered by the fact that only relatively small amounts of the enzyme could be extracted from E coli.
Their work was helped by the recent installation of a fermentor in the department for the growth of E coli which supplied hundreds of grams of log phase E coli.
With the aid of chromatography Kornberg's team was able to obtain a several thousand-fold purified but not yet homogeneous preparation of DNA polymerase.
While still impure, the enzyme proved capable of DNA replication. Many other DNA polymerases have been isolated from E coli since the s, two of them identified by Kornberg's son, Thomas. DNA polymerases have also been purified from other bacteria. This includes Taq DNA polymerase purified from the bacterium Thermus aquaticus in which was found to live in the hot springs of Yellowstone Park in Wyoming by Thomas Brock in The advantage of Taq is that it can withstand very high temperatures.
This makes it suitable for use in PCR. Polymerase does not create a novel DNA strand from scratch. The names of DNA polymerases vary, depending on the domains. Only DNA polymerases with in vitro activity, if applicable, are shown. This enzyme has the typical amino acid sequence of the archaeal family B enzymes, but it showed a high extension rate while maintaining high fidelity, and therefore, the commercial product, KOD DNA polymerase KOD Pol , was developed and became popular as a PCR enzyme.
The underlying reason why this family B enzyme shows high extension speed is interesting. Comparisons of the crystallographic structures and amino acid sequences of KOD Pol with other archaeal family B enzymes revealed the logical explanation for the efficient extension ability of this enzyme. Many basic residues are located around the active site in the finger domain of KOD Pol. In addition, many Arg residues are located at the forked point, which is the predicted as the junction of the template binding region and the editing cleft.
Research on DNA polymerases in hyperthermophilic archaea is motivated by not only industrial applications, but also basic molecular biology, to elucidate the molecular mechanisms of genetic information processing systems at extremely hot temperatures. To identify all of the DNA polymerases in the archaeal cell, we tried to separate the DNA polymerase activities in the total cell extract of P. Three major fractions showed nucleotide incorporation activity after anion exchange column chromatography Resource Q column, GE Healthcare; Imamura et al.
In addition to the further purification of each fraction, the screening of the DNA polymerase activity from the heat-stable protein library, made from E. This was the first report of a eukaryotic-like initiator protein for DNA replication in Archaea. After the discovery of this DNA polymerase, the total genome sequence of Methanococcus jannaschii was published as the first complete archaeal genome Bult et al.
The two genes were not present in tandem, but were located separately on the genome. We cloned and expressed them in E. Three more total genome sequences were subsequently reported, and the genes for DP1 and DP2 were found in all them. Due to the lack of sequence homology to other DNA polymerases, we proposed a new family, family D, for this enzyme Cann and Ishino, Physical map of the P.
In parallel to the identification of DNA polymerase activities in the cell extract of P. By using a set of mixed primers based on the conserved sequences of motifs A and C in the family B DNA polymerase, a single band was amplified.
However, two different fragments were found after the cloning and sequencing of the PCR product. The full-length sequences of both pol -like genes were cloned from the P. Both of the gene products exhibited the heat stable DNA polymerase activity Uemori et al. Unfortunately, the performance of these two enzymes in PCR was not better than Pfu polymerase, and we discontinued further research on them. However, this was the first report that an archaeal cell has two different family B DNA polymerases.
In the early stages of the total genome sequences, all sequences were from Euryarchaeota Archaeoglobus fulgidus, Methanothermobacter thermautotrophicus, Pyrococcus horikoshii and the determination of the genome sequence of a crenarchaeal organism was delayed until that of A.
Taken together with the new knowledge at that time, it was predicted that euryarchaeal organisms have one DNA polymerase each from family B and family D, respectively, and crenarchaeal organisms have at least two family B enzymes in the cell. This overview of the distribution of DNA polymerases in Archaea is generally correct as shown in Figure 4 , which displays DNA polymerases in the archaeal phyla subdomains including newly proposed phyla from recent ecological research.
DNA polymerases in Archaea. The evolutionary relationships of six phyla in the domain Archaea are schematically shown with the DNA polymerases encoded in their genomes.
The family B DNA polymerases from extrachromosomal elements were excluded. All of the original biochemical data for P. However, PolD has not been commercially developed. At the early stage, hot start PCR was one of the big improvements for the specific amplification. This hot start PCR method is generally effective to prevent non-specific amplification. For this purpose, another idea was tested. A chemical modification of Taq polymerase inactivated its enzymatic activity at low temperatures, but the modification can be released by high temperature resulting in activation of Taq polymerase to start PCR.
This temperature-dependent reversible modification of the Taq protein led to the commercial product, AmpliTaq Gold, as the hot start PCR enzyme. Taq polymerase is a family A enzyme, and is applicable to practical dideoxy sequencing. However, the output of the sequencing data was not ideal as compared with that from T7 DNA polymerase known commercially as Sequenase; see below.
An ingenious protein engineering strategy produced a mutant Taq polymerase that is more suitable for dideoxy sequencing than the wild type Taq polymerase. For this property, the strength of each signal is not uniform, but is distinctly unbalanced. However, T7 DNA polymerase equally incorporates deoxynucleotides and dideoxynucleotides, and therefore, it is easy to adjust the reaction conditions to provide very clear signals Tabor and Richardson, A detailed comparison of E.
This work was applied to Taq polymerase and a modified Taq with FY, which endows Taq with T7-type substrate recognition, was created Tabor and Richardson, This enzyme was called Thermosequenase, and it became popular as the standard enzyme for the fluorescently labeled sequencing method Reeve and Fuller, Another target for the creation of a new enzyme by mutagenesis is an enzyme that is more resistant to PCR inhibitors in blood or soil, such as hemoglobin and humic acid.
A mutant Taq DNA polymerase with enhanced resistance to various inhibitors, including whole blood, plasma, hemoglobin, lactoferrin, serum IgG, soil extracts, and humic acid, was successfully created by site-directed mutagenesis Kermekchiev et al. Furthermore, enzymes with a broad substrate specificity spectrum, which are thus useful for the amplification of ancient DNA containing numerous lesions, were also obtained by the CSR technique Ghadessy et al.
HhH is a widespread motif and generally functions on sequence-nonspecific DNA binding. These hybrid enzymes increased thermostability and became more resistant to salt and several inhibitors such as phenol, blood, and DNA intercalating dyes Pavlov et al. This enzyme shows very high processivity and accurate PCR performance, and is now widely used.
Another idea to improve the processivity of the archaeal family B DNA polymerases was to use PCNA proliferating cell nuclear antigen as a processivity factor. Originally, we determined the crystal structure of P.
Mutations of the amino acid residues involved in the ion pairs clearly decreased its ring stability, but unexpectedly, a less stable mutant PfuPCNA enhanced the primer extension reaction of Pfu DNA polymerase in vitro Matsumiya et al. Because of the high sensitivity of PCR, very small amounts of carry-over contaminants from previous PCRs are considered to be one of the major sources of false positive results. One problem of the archaeal family B DNA polymerase to be used for this carry-over prevention is that they specifically interact with uracil and hypoxanthine, which stalls their progression on DNA template strands Connolly, The crystal structure of the DNA polymerase revealed that read-ahead recognition occurs by an interaction with the deaminated bases in an N-terminal binding pocket that is specifically found in the archaeal family B DNA polymerases Fogg et al.
To conquer this defect, a point mutation V98Q was introduced into Pfu polymerase. This mutant enzyme is completely unable to recognize uracil, while its DNA polymerase activity is unaffected Fogg et al.
Therefore, this mutant Pfu polymerase is useful for the carry-over prevention PCR. Polymerase chain reaction initiated a revolution in molecular biology, and is now used daily not only in research, but also in the general human society. Notably, an enzyme with faster, longer, and more efficient extension ability, as compared to the properties of the current commercial products, will contribute to further improvements in PCR technology.
In addition to these basic abilities, DNA polymerases that can incorporate various modified nucleotides, which are useful for highly sensitive labeling, are valuable for single molecule analysis. Mutations of the DNA polymerase itself, by site-specific or random mutagenesis, are effective ways to create modified enzymes with improved PCR performance or specific properties for in vitro DNA manipulations. An artificial evolution procedure also has attracted a great deal of attention, for the creation of DNA polymerases with novel activities Brakmann, ; Henry and Romesberg, ; Holmberg et al.
Our strategy of using environmental DNA as a genetic resource also works well to investigate the structure—function relationships of DNA polymerases. The region corresponding to the active center of the DNA polymerizing reaction, in the structural genes of Taq polymerase and Pfu polymerase, was substituted with PCR fragments amplified from DNAs within soil samples from various locations in Japan.
The chimeric pol genes were constructed within the expression plasmids for the Taq and Pfu polymerases in E. The chimeric enzymes thus produced, exhibited DNA polymerase activities with different properties Matsukawa et al.
The main focus for the future development of DNA polymerases is not on versatile enzymes, but rather on specialized enzymes suitable for individual purposes, including whole genome amplification, rapid detection of short DNA, new sequencing technologies, etc.
Continued research on DNA polymerases may facilitate the invention of new genetic analysis technologies that are completely different from PCR or PCR-related techniques. The isothermal amplification without temperature cycling is more convenient and practical than PCR, and development of this type of technique has been actively performed Gill and Ghaemi, Several methods practically utilized now are based on the strand displacement SD activity of the DNA polymerases.
Alternatively, helicase was applied for the dissociation of the double-stranded DNA from an idea to mimic DNA replication in vivo Vincent et al. Although the helicase-dependent amplification HDA technique has not been practically used Jeong et al.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The writing of this review article was supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan grant number to Yoshizumi Ishino. Baar, C. Molecular breeding of polymerases for resistance to environmental inhibitors. Nucleic Acids Res. Barns, W. PCR amplification of up to kb DNA with high fidelity and high yield from lambda bacteriophage templates.
Brakmann, S. Directed evolution as a tool for understanding and optimizing nucleic acid polymerase function. Life Sci. Bult, C. Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii. Science , — CrossRef Full Text. Cann, I. Two family B DNA polymerases in Aeropyrum pernix , an obligate aerobic hyperthermophilic crenarchaeote. Archaeal DNA replication: identifying the pieces to solve a puzzle. Genetics , — Pubmed Abstract Pubmed Full Text.
Cariello, N. Chien, A. Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. Connolly, B. Recognition of deaminated bases by archaeal family-B DNA polymerases. Molecular breeding of polymerases for amplification of ancient DNA. Diaz, R. Accuracy of replication in the polymerase chain reaction.
Firbank, S. Uracil recognition in archaeal DNA polymerases captured by X-ray crystallography. Fogg, M. Structural basis for uracil recognition by archaeal family B DNA polymerases.
Forterre, P. Aphidicolin inhibits growth and DNA synthesis in halophilic archaebacteria. Ghadessy, F. Directed evolution of polymerase function by compartmentalized self-replication. Generic expansion of the substrate spectrum of a DNA polymerase by directed evolution. Gill, P. Nucleic acid isothermal amplification technologies.
Nucleosides Nucleotides Nucleic Acids 27, — Gray, M. The third form of life. Nature Greenough, L. Extremophiles 18, — Hashimoto, H. Nishioka, M. Henry, A. The evolution of DNA polymerases with novel activities. Holmberg, R.
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