Site-directed chemical modification of archaeal Thermococcus litoralis Sh1B DNA polymerase: Acquired ability to read through template-strand uracils

2010 ◽  
Vol 1804 (6) ◽  
pp. 1385-1393 ◽  
Author(s):  
Edita Gaidamaviciute ◽  
Daiva Tauraite ◽  
Julius Gagilas ◽  
Arunas Lagunavicius
Keyword(s):  
Chemical Modification ◽  
Dna Polymerase ◽  
Thermococcus Litoralis ◽  
Template Strand

Optimal conditions and specific characteristics of Vent exo– DNA polymerase in ligation-mediated polymerase chain reaction protocols

2005 ◽  
Vol 83 (2) ◽  
pp. 147-165 ◽  
Author(s):  
François Vigneault ◽  
Régen Drouin
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Polymerase ◽  
Pyrococcus Furiosus ◽  
Pcr Amplification ◽  
Polymerase Activity ◽  
Optimal Dosage ◽  
Chain Reaction ◽  
Thermus Aquaticus ◽  
Thermococcus Litoralis ◽  
Polymerase Chain

An optimized procedure for the ligation-mediated polymerase chain reaction (PCR) technique using Thermococcus litoralis exo– DNA polymerase (Vent exo–) was developed. The optimal dosage of Vent exo– at the primer extension and PCR amplification steps as well as the optimal DNA quantity to use were established. We showed that Vent exo– can efficiently create the blunt-ended termini required for subsequent linker ligation. Vent exo– proves to be more efficient than Pyrococcus furiosus exo– (Pfu exo–) for this task. Vent exo– resolves highly GC-rich sequence substantially better than Thermus aquaticus DNA polymerase (Taq) and with a similar efficiency as Pfu exo–. The DNA/DNA polymerase activity ratio is significantly higher for Vent exo– than for Pfu exo–, which is reflected by the sensibility of Vent exo– in efficiently amplifying genomic DNA. Furthermore, the range of efficiency of Vent exo– demonstrates the importance of conducting evaluative testing to identify the optimal dosage of use of this polymerase to obtain successful PCR amplification. Optimal MgSO4 concentrations to use with Vent exo– were established. Our results show that Vent exo– DNA polymerase produces bands of uniform and strong intensity and can efficiently be used for the analysis of DNA in living cells by ligation-mediated PCR.Key words: Vent exo– DNA polymerase, Pfu exo– DNA polymerase, DNA sequence context, ligation-mediated polymerase chain reaction (PCR), PCR buffer.

scholarly journals DNA Helicase–Polymerase Coupling in Bacteriophage DNA Replication

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1739
Author(s):  
Chen-Yu Lo ◽  
Yang Gao
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Molecular Mechanisms ◽  
Bacteriophage T4 ◽  
Dna Helicase ◽  
Model Systems ◽  
Genome Replication ◽  
Template Strand ◽  
Replication Systems ◽  
Mechanistic Basis

Bacteriophages have long been model systems to study the molecular mechanisms of DNA replication. During DNA replication, a DNA helicase and a DNA polymerase cooperatively unwind the parental DNA. By surveying recent data from three bacteriophage replication systems, we summarized the mechanistic basis of DNA replication by helicases and polymerases. Kinetic data have suggested that a polymerase or a helicase alone is a passive motor that is sensitive to the base-pairing energy of the DNA. When coupled together, the helicase–polymerase complex is able to unwind DNA actively. In bacteriophage T7, helicase and polymerase reside right at the replication fork where the parental DNA is separated into two daughter strands. The two motors pull the two daughter strands to opposite directions, while the polymerase provides a separation pin to split the fork. Although independently evolved and containing different replisome components, bacteriophage T4 replisome shares mechanistic features of Hel–Pol coupling that are similar to T7. Interestingly, in bacteriophages with a limited size of genome like Φ29, DNA polymerase itself can form a tunnel-like structure, which encircles the DNA template strand and facilitates strand displacement synthesis in the absence of a helicase. Studies on bacteriophage replication provide implications for the more complicated replication systems in bacteria, archaeal, and eukaryotic systems, as well as the RNA genome replication in RNA viruses.

scholarly journals Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase—an extremely heat stable enzyme with proofreading activity

1991 ◽  
Vol 19 (18) ◽  
pp. 4967-4973 ◽  
Author(s):  
P. Mattila ◽  
J. Korpela ◽  
T. Tenkanen ◽  
K. Pitkämem
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Dna Sequences ◽  
Total Concentration ◽  
Dna Amplification ◽  
Base Substitution ◽  
Thermococcus Litoralis ◽  
Heat Stable ◽  
Original Target

Abstract We demonstrate that the DNA polymerase isolated from Thermococcus litoralis (VentTM DNA polymerase) is the first thermostable DNA polymerase reported having a 3′—5′ proofreading exonuclease activity. This facilitates a highly accurate DNA synthesis in vitro by the polymerase. Mutational frequencies observed in the base substitution fidelity assays were in the range of 30×10−6. These values were 5–10 times lower compared to other thermostable DNA polymerases lacking the proofreading activity. All classes of DNA polymerase errors (transitions, transversions, frameshift mutations) were assayed using the forward mutational assay (1). The mutation frequencies of Thermococcus litoralis DNA polymerase varied between 15−35×10−4 being 2 – 4 times lower than the respective values obtained using enzymes without proofreading activity. We also noticed that the fidelity of the DNA polymerase from Thermococcus litoralis responds to changes in dNTP concentration, units of enzyme used per one reaction and the concentration of MgSO4 relative to the total concentration of dNTPs present in the reaction. The high fidelity DNA synthesis In vitro by Thermococcus litoralis DNA polymerase provides good possibilities for maintaining the genetic information of original target DNA sequences intact in the DNA amplification applications.

scholarly journals Chemical modification of aphidicolin and the inhibitory effects of its derivatives on DNA polymerase .ALPHA. in vitro.

1987 ◽  
Vol 35 (4) ◽  
pp. 1641-1644 ◽  
Author(s):  
Sayoko Hiranuma ◽  
Takeshi Shimizu ◽  
Hirosuke Yoshioka ◽  
Katsuhiko Ono ◽  
Hideo Nakane ◽  
...  
Keyword(s):  
Chemical Modification ◽  
Dna Polymerase ◽  
Inhibitory Effects ◽  
Dna Polymerase Alpha
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