Compartmentalized self-replication (CSR) selection of Thermococcus litoralis Sh1B DNA polymerase for diminished uracil binding

2010 ◽  
Vol 23 (8) ◽  
pp. 589-597 ◽  
Author(s):  
Agne Tubeleviciute ◽  
Remigijus Skirgaila
2010 ◽  
Vol 2010 ◽  
pp. 1-15 ◽  
Author(s):  
Richard G. Federley ◽  
Louis J. Romano

DNA replication is vital for an organism to proliferate and lying at the heart of this process is the enzyme DNA polymerase. Most DNA polymerases have a similar three dimensional fold, akin to a human right hand, despite differences in sequence homology. This structural homology would predict a relatively unvarying mechanism for DNA synthesis yet various polymerases exhibit markedly different properties on similar substrates, indicative of each type of polymerase being prescribed to a specific role in DNA replication. Several key conformational steps, discrete states, and structural moieties have been identified that contribute to the array of properties the polymerases exhibit. The ability of carcinogenic adducts to interfere with conformational processes by directly interacting with the protein explicates the mutagenic consequences these adducts impose. Recent studies have identified novel states that have been hypothesised to test the fit of the nascent base pair, and have also shown the enzyme to possess a lively quality by continually sampling various conformations. This review focuses on the homologous structural changes that take place in various DNA polymerases, both replicative and those involved in adduct bypass, the role these changes play in selection of a correct substrate, and how the presence of bulky carcinogenic adducts affects these changes.


2004 ◽  
Vol 284 (1-2) ◽  
pp. 147-157 ◽  
Author(s):  
Myung-Shin Lee ◽  
Myung-Hee Kwon ◽  
Kyongmin Hwang Kim ◽  
Ho-Joon Shin ◽  
Sun Park ◽  
...  

2005 ◽  
Vol 83 (2) ◽  
pp. 147-165 ◽  
Author(s):  
François Vigneault ◽  
Régen Drouin

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.


2016 ◽  
Author(s):  
Lars Witting

AbstractInter-specific body mass allometries can evolve from the natural selection of mass, with ±1/4 and ±3/4 exponents following from the geometry of intra-specific interactions when density dependent foraging occurs in two spatial dimensions (2D, Witting, 1995). The corresponding values for three dimensional interactions (3D) are ±1/6 and ±5/6.But the allometric exponents in mobile organisms are more diverse than the prediction. The exponent for mass specific metabolism tends to cluster around −1/4 and −1/6 in terrestrial and pelagic vertebrates, but it is strongly positive in prokaryotes with an apparent value around 5/6 (DeLong et al., 2010). And a value around zero has been reported in protozoa, and on the macro evolutionary scale from prokaryotes over larger unicells to multicellular vertebrates (Makarieva et al., 2005, 2008).I show that mass specific metabolism can be selected as the pace of the resource handling that generates net energy for self-replication and the selection of mass, and that this selection of metabolism and mass is sufficient to explain metabolic exponents that decline from 5/6 over zero to −1/6 in 3D, and from 3/4 over zero to −1/4 in 2D. The decline follows from a decline in the importance of mass specific metabolism for the selection of mass, and it suggestsi) that the body mass variation in prokaryotes is selected from primary variation in mass specific metabolism,ii) that the variation in multicellular animals are selected from primary variation in the handling and/or densities of the underlying resources,iii) that protozoa are selected as an intermediate lifeform between prokaryotes and multicellular animals, andiv) that macro evolution proceeds along an upper bound on mass specific metabolism.


2016 ◽  
Vol 6 (4) ◽  
pp. 407-410
Author(s):  
Inna B. Chastukhina ◽  
Liliia R. Nigmatullina ◽  
Lia R. Valeeva ◽  
Eugene V. Shakirov

2000 ◽  
Vol 46 (5) ◽  
pp. 620-624 ◽  
Author(s):  
Caroline J Fuery ◽  
Helen L Impey ◽  
Natalie J Roberts ◽  
Tanya L Applegate ◽  
Robyn L Ward ◽  
...  

Abstract Background: Restriction endonuclease-mediated selective (REMS)-PCR, allows detection of point mutations, deletions, and insertions. Reactions require concurrent activity of a restriction endonuclease (RE) and a DNA polymerase, both of which must be sufficiently thermostable to retain activity during thermocycling. The inclusion of the RE in REMS-PCR inhibits amplification of sequences containing the RE recognition site, thus producing selective amplification of sequences that lack the RE site. Methods: Assays were used that allowed the selection of conditions that produce concurrent RE/DNA polymerase activity. The RE thermostability assay involved thermocycling a RE under various conditions and assessing residual cleavage activity at various time points. Conditions found to preserve RE activity during thermocyling were then tested for their compatibility with DNA polymerase-mediated PCR. Results: A range of conditions that preserve activity of the RE BstNI over 30 cycles of PCR was identified. A subset of these conditions was subsequently found to mediate specific amplification using Taq DNA polymerase. These conditions were used to develop a REMS-PCR protocol for the detection of mutations at codon 12 of the K-ras gene. This protocol allowed the detection of 1 mutant allele in a background of 1000 wild-type alleles. The presence of primer sets for RE and PCR control amplicons provided unambiguous assessment of mutant status. Conclusion: Implementation of the assays described may facilitate development of REMS-PCR assays targeted to other loci associated with disease.


2014 ◽  
Vol 70 (a1) ◽  
pp. C200-C200
Author(s):  
Matthew Hogg ◽  
Pia Osterman ◽  
Göran Bylund ◽  
Rais Ganai ◽  
Else-Britt Lundström ◽  
...  

DNA polymerase ε (Pol ε) is a high-fidelity polymerase that participates in leading-strand synthesis during eukaryotic DNA replication in eukaryotic cells. The 2.2 Å ternary structure of the 142 kDa catalytic core of Pol ε from Saccharomyces cerevisiae in complex with DNA and an incoming nucleotide has recently been determined [1]. The structure provides information about the selection of the correct nucleotide and the positions of amino acids that might be critical for proofreading activity. Pol ε has the highest fidelity among B-family polymerases despite the absence of an extended β-hairpin loop that is required for high-fidelity replication by other B-family polymerases. Moreover, the catalytic core has a new domain (i.e. the P-domain) that allows Pol ε to encircle the nascent double-stranded DNA and enhance processifivity of the polymerase. The structure provides valuable insights into the similarities and differences between Pol ε and other B-family polymerases, and suggests possible mechanisms responsible for the high processivity and fidelity of Pol ε.


2018 ◽  
Vol 10 (1) ◽  
pp. 1-17 ◽  
Author(s):  
Zhanar Abil ◽  
Andrew D. Ellington

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