Addition of hydroxyl radicals to the C8 position of 2 0-deoxyguanosine generates an 8-hydroxyguanyl radical that can be converted into either 8-oxo-7,8-dihydro-2 0-deoxyguanosine or N-(2-deoxy-D-pento-furanosyl)-N-(2,6-diamino-4-hydroxy-5-formamido-pyrimidine) (Fapy-dG). The Fapy-dG adduct can adopt different conformations and in particular, can exist in an unnatural a anomeric configuration in addition to canonical b configuration. Previous studies reported that in 5 0-TGN-3 0 sequences, Fapy-dG predominantly induced G ! T transversions in both mammalian cells and Escherichia coli, suggesting that mutations could be formed either via insertion of a dA opposite the 5 0 dT due to primer/template mis-alignment or as result of direct miscoding. To address this question, single-stranded vectors containing a site-specific Fapy-dG adduct were generated to vary the identity of the 5 0 nucleotide. Following vector replication in primate cells (COS7), complex mutation spectra were observed that included $3-5% G ! T transversions and $14-21% G ! A transitions. There was no correlation apparent between the identity of the 5 0 nucleotide and spectra of mutations. When conditions for vector preparation were modified to favor the b anomer, frequencies of both G ! T and G ! A substitutions were significantly reduced. Mutation frequencies in wild-type E. coli and a mutant deficient in damage-inducible DNA polymerases were significantly lower than detected in COS7 and spectra were dominated by deletions. Thus, mutagenic bypass of Fapy-dG can proceed via mechanisms that are different from the previously proposed primer/template misalignment or direct misinser-tions of dA or dT opposite to the b anomer of Fapy-dG. Environ. Mol. Mutagen. 58:182-189, 2017. V C 2017 Wiley Periodicals, Inc.

The rapidly growing supplement industry operates without a formal premarket approval process. Consumers rely on product labels to be accurate and true. Those products containing live microbials report both identity and viability on most product labels. This study used next-generation sequencing technology as an analytical tool in conjunction with classic culture methods to examine the validity of the labels on supplement products containing live microbials found in the United States marketplace. Our results show the importance of testing these products for identity, viability, and potential contaminants, as well as introduce a new culture-independent diagnostic approach for testing these products.

The formamidopyrimidines Fapy.dA and Fapy.dG are produced in DNA as a result of oxidative stress. These lesions readily epimerize in water, an unusual property for nucleosides. The equilibrium mixture slightly favors the beta-anomer, but the configurational status in DNA is unknown. The ability of endonuclease IV (Endo IV) to efficiently incise alpha-deoxyadenosine was used as a tool to determine the configuration of Fapy.dA and Fapy.dG in DNA. Endo IV incision of the C-nucleoside analogues of Fapy.dA was used to establish selectivity for the alpha-anomer. Incision of alpha-C-Fapy.dA follows Michaelis-Menten kinetics (K(m) = 144.0 +/- 7.5 nM, k(cat) = 0.58 +/- 0.21 min(-1)), but the beta-isomer is a poor substrate. Fapy.dA incision is considerably slower than that of alpha-C-Fapy.dA, and does not proceed to completion. Endo IV incision of Fapy.dA proceeds further upon rehybridization, suggesting that the lesion reequilibrates and that the enzyme preferentially cleaves duplex DNA containing alpha-Fapy.dA. The extent of Fapy.dA incision suggests that the lesion exists predominantly ( approximately 90%) as the beta-anomer in DNA. Endo IV incises Fapy.dG to less than 5% under comparable reaction conditions, suggesting that the lesion exists almost exclusively as its beta-anomer in DNA.

The nucleoside triphosphates of N6-(2-deoxy-alpha,beta-d-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy.dGTP) and its C-nucleoside analogue (beta-C-Fapy.dGTP) were synthesized. The lability of the formamide group required that nucleoside triphosphate formation be carried out using an umpolung strategy in which pyrophosphate was activated toward nucleophilic attack. The Klenow fragment of DNA polymerase I from Escherichia coli accepted Fapy.dGTP and beta-C-Fapy.dGTP as substrates much less efficiently than it did dGTP. Subsequent extension of a primer containing either modified nucleotide was less affected compared to when the native nucleotide is present at the 3'-terminus. The specificity constants are sufficiently large that nucleoside triphosphate incorporation could account for the level of Fapy.dG observed in cells if 1% of the dGTP pool is converted to Fapy.dGTP. Similarly, polymerase-mediated introduction of beta-C-Fapy.dG could be useful for incorporating useful amounts of this nonhydrolyzable analogue for use as an inhibitor of base excision repair. The kinetic viability of these processes is enhanced by inefficient hydrolysis of Fapy.dGTP and beta-C-Fapy.dGTP by MutT, the E. coli enzyme that releases pyrophosphate and the corresponding nucleoside monophosphate upon reaction with structurally related nucleoside triphosphates.

Fapy.dG (N(6)()-(2-deoxy-alpha,beta-d-erythropentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine) is a modified purine lesion produced by a variety of DNA-damaging agents, which shows interesting biochemical properties. The previous method for synthesizing oligonucleotides containing Fapy.dG utilized a reverse dinucleotide phosphoramidite, which also required the synthesis of the appropriate reverse phosphoramidites. An improved method for synthesizing oligonucleotides containing Fapy.dG, which does not require reverse phosphoramidites, is described. Fapy.dG containing dinucleotide phosphoramidites containing 5'-thymidine (11a) or 5'-deoxycytidine (15) are prepared and employed in oligonucleotide synthesis. Oligonucleotide purity is assayed using the DNA repair enzyme formamidopyrimidine DNA glycosylase and by ESI-MS.

We used a series of dNTP analogues in conjunction with templates containing modified bases to elucidate the role that N 2 of a purine plays during dNTP polymerization by human DNA polymerase α. Removing N 2 from dGTP had small effects during correct incorporation opposite C, but specifically increased misincorporation opposite A. Adding N 2 to dATP and related analogues had small and variable effects on the efficiency of polymerization opposite T. However, the presence of N 2 greatly enhanced polymerization of these dATP analogues opposite a template C. The ability of N 2 to enhance polymerization opposite C likely results from formation of a hydrogen bond between the purine N 2 and pyrimidine O 2 . Even in those cases where formation of a wobble base-pair, tautomerization, and/or protonation of the base-pair between the incoming dNTP and template base cannot occur (Eg., 2-pyridone:purine (or purine analogue) base-pairs), N 2 enhanced formation of the base-pair. Importantly, N 2 had similar effects on dNTP polymerization both when added to the incoming purine dNTP and the template base being replicated. The mechanistic implications of these results regarding how pol α discriminates between right and wrong dNTPs are discussed. KeywordsFidelity; misincorporation; base-pair; nucleotide; dITP Accurate DNA replication is crucial for cell survival. Fortunately, most replicative DNA polymerases rarely misincorporate dNTPs, exhibiting typical error rates of 10 −3 -10 −6 errors per nucleotide replicated (3). When misincorporation does occur, the rate of elongation decreases substantially, thereby allowing for exonucleolytic proofreading by a 3'−5' exonuclease (4). Presently, the specific mechanisms different polymerases employ to distinguish between right and wrong dNTP substrates during the polymerization reaction remain unclear. Structural and biophysical analysis of various polymerases has suggested that this polymerase differentiates between substrates by the opening and closing of the polymerase . When the complex is in an open conformation, the active site of the enzyme becomes solvent accessible and allows the polymerase to test its dNTP substrates. After initial binding of a dNTP, the mechanism employed for dNTP selection, however, remains a topic of debate. One model posits that Watson-Crick hydrogen bonding between the incoming dNTP and the template base provides the driving force for the incorporation of correct dNTPs. Consistent with this model, the low fidelity enzymes human primase and herpes primase, only efficiently †

To better understand how DNA polymerases interact with mutagenic bases, we examined how human DNA polymerase α (pol α), a B family enzyme, and DNA polymerase from Bacillus stearothermophilus (BF), an A family enzyme, generate adenine:hypoxanthine and adenine:8-oxo-7,8-dihydroguanine (8-oxoG) base pairs. Pol α strongly discriminated against polymerizing dATP opposite 8-oxoG, and removing N1, N 6 , or N7 further inhibited incorporation, whereas removing N3 from dATP dramatically increased incorporation (32-fold). Eliminating N 6 from 3-deaza-dATP now greatly reduced incorporation, suggesting that incorporation of dATP (analogues) opposite 8-oxoguanine proceeds via a Hoogsteen base-pair and that pol α uses N3 of a purine dNTP to block this incorporation. Pol α also polymerized 8-oxo-dGTP across from a templating A, and removing N 6 from the template adenine inhibited incorporation of 8-oxoG. The effects of N1, N 6 , and N7 demonstrated a strong interdependence during formation of adenine:hypoxanthine base-pairs by pol α and N3 of dATP again helps prevent polymerization opposite a templating hypoxanthine. BF very efficiently polymerized 8-oxo-dGTP opposite adenine, and N1 and N7 of adenine appear to play important roles. BF incorporates dATP opposite 8-oxoG less efficiently, and modifying N1, N 6 , or N7 greatly inhibits incorporation. N 6 , and to a lesser extent N1, help drive hypoxanthine:adenine base pair formation by BF. The mechanistic implications of these results showing that different polymerases interact very differently with base lesions are discussed.

To better understand the energetics of accurate DNA replication, we directly measured ΔGO for the incorporation of a nucleotide into elongating dsDNA in solution (ΔGOincorporation). Direct measurements of the energetic difference between synthesis of correct and incorrect base pairs found it to be much larger than previously believed (average ΔΔGOincorporation = 5.2±1.34 kcal mol−). Importantly, these direct measurements indicate that ΔΔGOincorporation alone can account for the energy required for highly accurate DNA replication. Evolutionarily, these results indicate that the earliest polymerases did not have to evolve sophisticated mechanisms to replicate nucleic acids, they may have only had to take advantage of the inherently more favorable ΔGO for polymerization of correct nucleotides. These results also provide a basis for understanding how polymerases replicate DNA (or RNA) with high fidelity.