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.