DNA Polymerase C of the Thermophilic Bacterium Thermus aquaticus: Classification and Phylogenetic Analysis of the Family C DNA Polymerases

1999 ◽  
Vol 48 (6) ◽  
pp. 756-769 ◽  
Author(s):  
Yi-Ping Huang ◽  
Junetsu Ito
Keyword(s):  
Phylogenetic Analysis ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Thermophilic Bacterium ◽  
Thermus Aquaticus ◽  
The Family

scholarly journals Isolation and Phylogenetic Analysis of Novel Viruses Infecting the Phytoplankton Phaeocystis globosa (Prymnesiophyceae)

2004 ◽  
Vol 70 (6) ◽  
pp. 3700-3705 ◽  
Author(s):  
C. P. D. Brussaard ◽  
S. M. Short ◽  
C. M. Frederickson ◽  
C. A. Suttle
Keyword(s):  
Phylogenetic Analysis ◽  
Dna Polymerase ◽  
Coastal Waters ◽  
Common Ancestor ◽  
Genetic Relatedness ◽  
Amino Acid Sequences ◽  
Polymerase Gene ◽  
Phaeocystis Globosa ◽  
The Family ◽  
Algal Host

ABSTRACT Viruses infecting the harmful bloom-causing alga Phaeocystis globosa (Prymnesiophyceae) were readily isolated from Dutch coastal waters (southern North Sea) in 2000 and 2001. Our data show a large increase in the abundance of putative P. globosa viruses during blooms of P. globosa, suggesting that viruses are an important source of mortality for this alga. In order to examine genetic relatedness among viruses infecting P. globosa and other phytoplankton, DNA polymerase gene (pol) fragments were amplified and the inferred amino acid sequences were phylogenetically analyzed. The results demonstrated that viruses infecting P. globosa formed a closely related monophyletic group within the family Phycodnaviridae, with at least 96.9% similarity to each other. The sequences grouped most closely with others from viruses that infect the prymnesiophyte algae Chrysochromulina brevifilum and Chrysochromulina strobilus. Whether the P. globosa viruses belong to the genus Prymnesiovirus or form a separate group needs further study. Our data suggest that, like their phytoplankton hosts, the Chrysochromulina and Phaeocystis viruses share a common ancestor and that these prymnesioviruses and their algal host have coevolved.

scholarly journals Capacity of Nine Thermostable DNA Polymerases To Mediate DNA Amplification in the Presence of PCR-Inhibiting Samples

1998 ◽  
Vol 64 (10) ◽  
pp. 3748-3753 ◽  
Author(s):  
Waleed Abu Al-Soud ◽  
Peter Rådström
Keyword(s):  
Dna Polymerase ◽  
Biological Samples ◽  
Detection Method ◽  
Dna Polymerases ◽  
Dna Amplification ◽  
Nucleic Acid Amplification ◽  
Full Potential ◽  
Thermus Aquaticus ◽  
Pcr Inhibitors ◽  
Thermostable Dna Polymerase

ABSTRACT The PCR is an extremely powerful method for detecting microorganisms. However, its full potential as a rapid detection method is limited by the inhibition of the thermostable DNA polymerase fromThermus aquaticus by many components found in complex biological samples. In this study, we have compared the effects of known PCR-inhibiting samples on nine thermostable DNA polymerases. Samples of blood, cheese, feces, and meat, as well as various ions, were added to PCR mixtures containing various thermostable DNA polymerases. The nucleic acid amplification capacity of the nine polymerases, under buffer conditions recommended by the manufacturers, was evaluated by using a PCR-based detection method for Listeria monocytogenes in the presence of purified template DNA and different concentrations of PCR inhibitors. The AmpliTaqGold and the Taq DNA polymerases from Thermus aquaticus were totally inhibited in the presence of 0.004% (vol/vol) blood in the PCR mixture, while the HotTub, Pwo, rTth, andTfl DNA polymerases were able to amplify DNA in the presence of 20% (vol/vol) blood without reduced amplification sensitivity. The DNA polymerase from Thermotoga maritima(Ultma) was found to be the most susceptible to PCR inhibitors present in cheese, feces, and meat samples. When the inhibitory effect of K and Na ions was tested on the nine polymerases, HotTub from Thermus flavus and rTthfrom Thermus thermophilus were the most resistant. Thus, the PCR-inhibiting effect of various components in biological samples can, to some extent, be eliminated by the use of the appropriate thermostable DNA polymerase.

scholarly journals yeast killer plasmid pGKL1 encodes a DNA polymerase belonging to the family B DNA polymerases

1987 ◽  
Vol 15 (21) ◽  
pp. 9088-9088 ◽  
Author(s):  
Guhung Jung ◽  
Mark C. Leavitt ◽  
Junetsu Ito
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerases ◽  
The Family ◽  
Killer Plasmid ◽  
Yeast Killer

scholarly journals Deoxynucleotide Triphosphate Binding Mode Conserved in Y Family DNA Polymerases

2003 ◽  
Vol 23 (8) ◽  
pp. 3008-3012 ◽  
Author(s):  
Robert E. Johnson ◽  
José Trincao ◽  
Aneel K. Aggarwal ◽  
Satya Prakash ◽  
Louise Prakash
Keyword(s):  
Dna Polymerase ◽  
Close Contact ◽  
Dna Polymerases ◽  
Binding Mode ◽  
Nucleotide Incorporation ◽  
The Family ◽  
And Function ◽  
High Degree ◽  
Y Family ◽  
Deoxynucleotide Triphosphate

ABSTRACT Although DNA polymerase η (Polη) and other Y family polymerases differ in sequence and function from classical DNA polymerases, they all share a similar right-handed architecture with the palm, fingers, and thumb domains. Here, we examine the role in Saccharomyces cerevisiae Polη of three conserved residues, tyrosine 64, arginine 67, and lysine 279, which come into close contact with the triphosphate moiety of the incoming nucleotide, in nucleotide incorporation. We find that mutational alteration of these residues reduces the efficiency of correct nucleotide incorporation very considerably. The high degree of conservation of these residues among the various Y family DNA polymerases suggests that these residues are also crucial for nucleotide incorporation in the other members of the family. Furthermore, we note that tyrosine 64 and arginine 67 are functionally equivalent to the deoxynucleotide triphosphate binding residues arginine 518 and histidine 506 in T7 DNA polymerase, respectively.

scholarly journals Duplication of a Truncated Paralog of the Family B DNA Polymerase Gene Aa-polB in the Agrocybe aegerita Mitochondrial Genome

2001 ◽  
Vol 67 (4) ◽  
pp. 1739-1743 ◽  
Author(s):  
Gerard Barroso ◽  
Frederic Bois ◽  
Jacques Labarère
Keyword(s):  
Mitochondrial Genome ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Polymerase Gene ◽  
Mitochondrial Trna ◽  
Amino Acid Similarity ◽  
Agrocybe Aegerita ◽  
Naturally Occurring ◽  
The Family ◽  
Dna Polymerase Gene

ABSTRACT The Agrocybe aegerita mitochondrial genome contains a truncated family B DNA polymerase gene (Aa-polB P1) whose nucleotide sequence is 86% identical to the previously described and potentially functional Aa-polB gene. A tRNAMetgene occurs at the 3′ end of the Aa-polB P1 gene. TheAa-polB P1 gene could result from reverse transcription of an Aa-polB mRNA primed by a tRNAMet followed by the integration of the cDNA after recombination at the mitochondrial tRNA locus. Two naturally occurring alleles of Aa-polB P1carry one or two copies of the disrupted sequence. In strains with two copies of Aa-polB P1, these copies are inverted relative to one another and separated by a short sequence carrying the tRNAMet gene. Both A. aegerita mitochondrial family B DNA polymerases were found to be related to other family B DNA polymerases (36 to 53% amino acid similarity), including the three enzymes of the archaebacterium Sulfolobus solfataricus. If mitochondria originated from a fusion between aClostridium-like eubacterium and aSulfolobus-like archaebacterium, then the A. aegerita family B DNA polymerase genes could be remnants of the archaebacterial genes.

scholarly journals Characterization of DNA Polymerase from Thermus thermophilus MAT72 Phage Tt72

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 38
Author(s):  
Sebastian Dorawa ◽  
Magdalena Plotka ◽  
Anna-Karina Kaczorowska ◽  
Olafur H. Fridjonsson ◽  
Gudmundur O. Hreggvidsson ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Thermus Thermophilus ◽  
Dna Polymerases ◽  
In Silico Analysis ◽  
Thermophilic Bacterium ◽  
Thermostable Enzymes ◽  
Reading Frame ◽  
E Coli ◽  
Gene Coding ◽  
Exonuclease Domain

Thermophilic phages are recognized as an untapped source of thermostable enzymes relevant in biotechnology; however, their biology is poorly explored. This has led us to start a project aimed at investigating thermophilic phages isolated from geothermal areas of Iceland. In this study, we present a structural and functional analysis of the DNA polymerase of phage Tt72, which infects thermophilic bacterium Thermus thermophilus MAT72. An in silico analysis of the Tt72 phage genome revealed the presence of a 2112-bp open reading frame (ORF) encoding protein homologous to the members of the A family of DNA polymerases. It contains a conserved nucleotidyltransferase domain and a 3′ → 5′ exonuclease domain but lacks the 5′ → 3′ exonuclease domain. The amino acid sequence of Tt72 DNA polymerase shows high similarity to two as yet uncharacterized DNA polymerases of T. thermophilus phages: ΦYS40 (91%) and ΦTMA (90%). The gene coding for Tt72 DNA polymerase was cloned and overexpressed in E. coli. The Tt72 polA gene is composed of 2112 nucleotides. The overall G+C content of this gene is 31.58%, which is lower than the G+C content of T. thermophilus genomic DNA (69.49%). The Tt72 polA gene codes for a 703-aa protein with a predicted molecular weight of 80,477. The enzyme was overproduced in E. coli, purified by heat treatment, followed by HiTrap TALON column and HiTrap Heparin HP column chromatography, then biochemically characterized. The optimum activity was found at 55 °C, pH 8.5, 25 mM KCl, and 0.5 mM Mg2+. Furthermore, the Tt72 DNA polymerase shows strong 3′ → 5′ exonucleolytic activity.

scholarly journals Structure of the family B DNA polymerase from the hyperthermophilic archaeonPyrobaculum calidifontis

2017 ◽  
Vol 73 (5) ◽  
pp. 420-427 ◽  
Author(s):  
Jingxu Guo ◽  
Wenling Zhang ◽  
Alun R. Coker ◽  
Steve P. Wood ◽  
Jonathan B. Cooper ◽  
...  
Keyword(s):  
Amino Acids ◽  
Dna Polymerase ◽  
Active Sites ◽  
Dna Polymerases ◽  
Structural Features ◽  
Salt Bridges ◽  
Central Channel ◽  
High Sequence Identity ◽  
Sequence Identity ◽  
The Family

The family B DNA polymerase fromPyrobaculum calidifontis(Pc-polymerase) consists of 783 amino acids and is magnesium-ion dependent. It has an optimal pH of 8.5, an optimal temperature of 75°C and a half-life of 4.5 h at 95°C, giving it greater thermostability than the widely usedTaqDNA polymerase. The enzyme is also capable of PCR-amplifying larger DNA fragments of up to 7.5 kb in length. It was shown to have functional, error-correcting 3′–5′ exonuclease activity, as do the related high-fidelity DNA polymerases fromPyrococcus furiosus,Thermococcus kodakarensisKOD1 andThermococcus gorgonarius, which have extensive commercial applications.Pc-polymerase has a quite low sequence identity of approximately 37% to these enzymes, which, in contrast, have very high sequence identity to each other, suggesting that theP. calidifontisenzyme is distinct. Here, the structure determination ofPc-polymerase is reported, which has been refined to anRfactor of 24.47% and anRfreeof 28.81% at 2.80 Å resolution. The domains of the enzyme are arranged in a circular fashion to form a disc with a narrow central channel. One face of the disc has a number of connected crevices in it, which allow the protein to bind duplex and single-stranded DNA. The central channel is thought to allow incoming nucleoside triphosphates to access the active site. The enzyme has a number of unique structural features which distinguish it from other archaeal DNA polymerases and may account for its high processivity. A model of the complex with the primer-template duplex of DNA indicates that the largest conformational change that occurs upon DNA binding is the movement of the thumb domain, which rotates by 7.6° and moves by 10.0 Å. The surface potential of the enzyme is dominated by acidic groups in the central region of the molecule, where catalytic magnesium ions bind at the polymerase and exonuclease active sites. The outer regions are richer in basic amino acids that presumably interact with the sugar-phosphate backbone of DNA. The large number of salt bridges may contribute to the high thermal stability of this enzyme.

scholarly journals Modification Of Taq DNA Polymerase with Improved Elongation Ability

2021 ◽  
Author(s):  
A. Sarsen ◽  
Zh. Akishev ◽  
M. Saginova ◽  
B. Sultankulov ◽  
B. Khassenov
Keyword(s):  
Dna Polymerase ◽  
Fusion Gene ◽  
Thermophilic Bacterium ◽  
Optimal Conditions ◽  
Thermus Aquaticus ◽  
T7 Promoter ◽  
Taq Polymerase ◽  
Taq Dna Polymerase ◽  
Diagnostic Practice ◽  
Polymerase I

Thermostable polymerases play a significant role in molecular biology and diagnostic practice. The most famous and demanded is Polymerase I from the thermophilic bacterium Thermus aquaticus (Taq-pol). This polymerase at one time made a kind of revolution in the polymerase chain reaction. In this work, we attempted to modify this polymerase by attaching an additional Sso7d protein from Sulfolobus solfataricus to Taq-pol, which provides additional binding to the double-stranded DNA of the template. Sso7d-Taq fusion gene was expressed in BL21(DE3) cells. Optimal conditions were selected for maximum production of modified Sso7d-Taq polymerase. The optimal conditions for the intracellular accumulation of Sso7d-Taq polymerase: activation of the T7 promoter when the optical density of the culture reaches OD600 = 0.8-1.0 by adding IPTG at a concentration of 0.2 mM, followed by incubation of the culture at 37°C for 20-24 hours. Recombinant Sso7d-Taq polymerase has been purified and tested by PCR for thermal stability and elongation time. It was found that the Sso7d-Taq enzyme withstands 5 hour incubation at 95°C and 75 minute incubation at 98°C. Comparative analysis with unmodified Taq DNA polymerase showed that the Sso7d-Taq enzyme reduces the elongation rate by several times - up to 15-13 seconds per 1 kbp. The results obtained indicate the prospects of using Sso7d-Taq DNA polymerase in scientific research and diagnostic practice.

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