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Purpose: To investigate the effect of the supplementation of energy drinks with green tea extract on abrasive and erosive dentin wear., Materials and Methods: Six groups, each comprising 15 bovine dentin samples, were prepared, yielding a total of 90 samples. Erosion was performed by immersing the samples in Red Bull and Red Bull light with and without green tea extract. Tap water with and without green tea extract was used as the control groups. The samples were subjected to abrasive/erosive cycling for five days. The following cycling was performed daily: toothbrush abrasion (20 brushstrokes; 2.5 N); eight erosive cycles (2 min storage in the respective solutions); in between the erosive cycles, storage in artificial saliva (60 min) and again toothbrush abrasion (20 brushstrokes; 2.5 N). During the night, samples were again stored in artificial saliva. Abrasive/erosive dentin wear was measured using a stylus profilometer (µm, accuracy = 40 nm). The measured dentin loss results from the vertical position shift on the y-axis from base to final profile after the wear process in 2D. Pairwise comparisons between the groups were carried out using Wilcoxon signed-rank test., Results: The following dentin wear (median [IQR]) was measured: Red Bull: 1.9 µm (0.5); Red Bull Light: 1.3 µm (0.3); Red Bull with green tea extract: 0.8 µm (0.3); Red Bull Light with green tea extract: 0.3 µm (0.5); Tap water with green tea extract: -0.2 µm (0.7); Tap water: -1.0 µm (1.2). The comparison of all tested groups to each other proved to be statistically significant (p < 0.05)., Conclusion: The supplementation of energy drinks with green tea extract provide a protective effect against erosive/abrasive wear in vitro.

The ability to detect DNA modification sites at single base resolution could significantly advance studies regarding DNA adduct levels, which are extremely difficult to determine. Artificial nucleotides that are specifically incorporated opposite a modified DNA site offer a potential strategy for detection of such sites by DNA polymerase-based systems. Here we investigate the action of newly synthesized base-modified benzimidazole-derived 2'-deoxynucleoside-5'-O-triphosphates on DNA polymerases when performing translesion DNA synthesis past the pro-mutagenic DNA adduct O(6)-benzylguanine (O(6)-BnG). We found that a mutated form of KlenTaq DNA polymerase, i.e., KTqM747K, catalyzed O(6)-BnG adduct-specific processing of the artificial BenziTP in favor of the natural dNTPs. Steady-state kinetic parameters revealed that KTqM747K catalysis of BenziTP is 25-fold more efficient for template O(6)-BnG than G, and 5-fold more efficient than natural dTMP misincorporation in adduct bypass. Furthermore, the nucleotide analogue BenziTP is required for full-length product formation in O(6)-BnG bypass, as without BenziTP the polymerase stalls at the adduct site. By combining the KTqM747K polymerase and BenziTP, a first round of DNA synthesis enabled subsequent amplification of Benzi-containing DNA. These results advance the development of technologies for detecting DNA adducts.

It is estimated that about 10,000 abasic sites are formed per day per cell. Abasic sites impose a significant challenge for bypass synthesis by DNA polymerases. Recently, a tyrosine in KlenTaq DNA polymerase has been highlighted as being crucial for nucleotide selection opposite abasic sites. Structural data indicated a hydrogen bond between the tyrosine's hydroxy group and the N3 of an incoming ddATP opposite the abasic site. In order to further investigate abasic site bypass, we incorporated the unnatural amino acid 2,3,5-trifluorotyrosine at the position of the crucial tyrosine of KlenTaq DNA polymerase. Fluorine substitution at the tyrosine decreased the pka value of the tyrosine's hydroxy group and allowed its protonation state to be modulated. Single-nucleotide-incorporation experiments revealed reduced activity for the KlenTaq mutant compared to the wild-type when bypassing an abasic site analogue. The finding stresses the involvement of this tyrosine and its hydrogen bonding in abasic site bypass., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

DNA is being constantly damaged by endo- and exogenous agents such as reactive oxygen species, chemicals, radioactivity, and ultraviolet radiation. Additionally, DNA is inherently labile, and this can result in, for example, the spontaneous hydrolysis of the glycosidic bond that connects the sugar and the nucleobase moieties in DNA; this results in abasic sites. It has long been obscure how cells achieve DNA synthesis past these lesions, and only recently has it been discovered that several specialized DNA polymerases are involved in translesion synthesis. The underlying mechanisms that render one DNA polymerase competent in translesion synthesis while another DNA polymerase fails are still indistinct. Recently two variants of Taq DNA polymerase that exhibited higher lesion bypass ability than the wild-type enzyme were identified by directed-evolution approaches. Strikingly, in both approaches it was independently found that substitution of a single nonpolar amino acid side chain by a cationic side chain increases the capability of translesion synthesis. Here, we combined both mutations in a single enzyme. We found that the KlenTaq DNA polymerase that bore both mutations superseded the wild-type as well as the respective single mutants in translesion-bypass proficiency. Further insights in the molecular basis of the detected gain of translesion-synthesis function were obtained by structural studies of DNA polymerase variants caught in processing canonical and damaged substrates. We found that increased positive charge of the surface potential in the area proximal to the negatively charged substrates promotes translesion synthesis by KlenTaq DNA polymerase, an enzyme that has very limited naturally evolved capability to perform translesion synthesis. Since expanded positively charged surface potential areas are also found in naturally evolved translesion DNA polymerases, our results underscore the impact of charge on the proficiency of naturally evolved translesion DNA polymerases., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Abasic sites represent the most frequent DNA lesions in the genome that have high mutagenic potential and lead to mutations commonly found in human cancers. Although these lesions are devoid of the genetic information, adenine is most efficiently inserted when abasic sites are bypassed by DNA polymerases, a phenomenon termed A-rule. In this study, we present X-ray structures of a DNA polymerase caught while incorporating a nucleotide opposite an abasic site. We found that a functionally important tyrosine side chain directs for nucleotide incorporation rather than DNA. It fills the vacant space of the absent template nucleobase and thereby mimics a pyrimidine nucleobase directing for preferential purine incorporation opposite abasic residues because of enhanced geometric fit to the active site. This amino acid templating mechanism was corroborated by switching to pyrimidine specificity because of mutation of the templating tyrosine into tryptophan. The tyrosine is located in motif B and highly conserved throughout evolution from bacteria to humans indicating a general amino acid templating mechanism for bypass of non-instructive lesions by DNA polymerases at least from this sequence family.