Identification of the Major Product of Guanine Oxidation in DNA by Ozone.

Ozonolysis of guanosine formed the 5-carboxamido-5-formamido-2-iminohydantoin (2Ih) nucleoside along with trace spiroiminodihydantoin (Sp). On the basis of literature precedent, we propose an unconventional ozone mechanism involving incorporation of only one oxygen atom of O3 to form 2Ih with evolution of singlet oxygen responsible for Sp formation. The increased yield of Sp in the buffered 1O2-stabilizing solvent D2O, formation of 2Ih in a short oligodeoxynucleotide, and 18O-isotope labeling provided evidence to support this mechanism. The elusiveness and challenges of working with 2Ih are described in a series of studies on the significant context effects on the half-life of the 2Ih glycosidic bond.

Insights into the 5-Carboxamido-5-Formamido-2-Iminohydantoin Structural Isomerization Equilibria.

Exposure of DNA to oxidants results in modification of the electron-rich guanine heterocycle including formation of the mutagenic 5-carboxamido-5-formamido-2-iminohydantoin (2Ih) lesion. Previously thought to exist solely as a pair of diastereomers, we found under biologically relevant conditions that 2Ih reversibly closes to a formerly hypothetical intermediate and opens into a newly discovered regioisomer. In a nucleoside model, only ∼80% of 2Ih existed as the structure studied over the last 20 years with significant isomeric products persisting in buffered aqueous solution.

Collateral Damage Occurs When Using Photosensitizer Probes to Detect or Modulate Nucleic Acid Modifications.

Nucleic acids are chemically modified to fine-tune their properties for biological function. Chemical tools for selective tagging of base modifications enables new approaches; the photosensitizers riboflavin and anthraquinone were previously proposed to oxidize N6 -methyladenine (m6 A) or 5-methylcytosine (5mdC) selectively. Herein, riboflavin, anthraquinone, or Rose Bengal were allowed to react with the canonical nucleosides dA, dC, dG, and dT, and the modified bases 5mdC, m6 A, 8-oxoguanine (dOG), and 8-oxoadenine (dOA) to rank their reactivities. The nucleoside studies reveal that dOG is the most reactive and that the native nucleoside dG is higher or similar in reactivity to 5mdC or m6 A; competition in both single- and double-stranded DNA of dG vs. 5mdC or 6mdA for oxidant confirmed that dG is favorably oxidized. Thus, photosensitizers are promiscuous nucleic acid oxidants with poor chemoselectivity that will negatively impact attempts at targeted oxidation of modified nucleotides in cells.

Simple kinetics, assay, and trends for soil microbial catalases.

In this report, we expand upon the enzymology and ecology of soil catalases through development and application of a simple kinetic model and field-amenable assay based upon volume displacement. Through this approach, we (A) directly relate apparent Michaelis-Menten terms to the catalase reaction mechanism, (B) obtain upper estimates of the intrinsic rate constants for the catalase community (k3'), along with moles of catalase per 16S rRNA gene copy number, (C) utilize catalase specific activities (SAs) to obtain biomass estimates of soil and permafrost communities (LOD, ~104 copy number gdw-1), and (D) relate kinetic trends to changes in bacterial community structure. In addition, this novel kinetic approach simultaneously incorporates barometric adjustments to afford comparisons across field measurements. As per our model, and when compared to garden soils, biological soil crusts exhibited ~2-fold lower values for k3', ≥105-fold higher catalase moles per biomass (250-1200 zmol copy number-1), and ~104-fold higher SAs per biomass (74-230 fkat copy number-1); whereas the highest SAs were obtained from permafrost and high-elevation soil communities (5900-6700 fkat copy number-1). In sum, the total trends suggest that microbial communities which experience higher degrees of native oxidative stress possess higher basal intracellular catalase concentrations and SAs per biomass.

Fluorophore-mediated Photooxidation of the Guanine Heterocycle.

Fluorescent dyes are routinely used to visualize DNA or RNA in various experiments, and some dyes also act as photosensitizers capable of catalyzing oxidation reactions. The present studies explored whether the common labeling dyes fluorescein, rhodamine, BODIPY, or cyanine3 (Cy3) can function as photosensitizers to oxidize nucleic acid polymers. Photoirradiation of each dye in the presence of the guanine (G) heterocycle, which is the most sensitive toward oxidation, identified slow rates of nucleobase oxidation in the nucleoside and DNA contexts. For all four fluorophores studied, the only product detected was spiroiminodihydantoin (Sp) suggesting the dyes functioned as Type II photosensitizers and generate singlet oxygen (1O2). The nucleoside reactions were then conducted in D2O solutions, known to increase the lifetime of 1O2, which resulted in a ~6-fold increase in the Sp yield, further supporting the classification of these dyes as Type II photosensitizers. Lastly, we inspected the pattern of G reactivity with the dyes upon photoirradiation in the context of a parallel-stranded G-quadruplex. The G nucleotides in the two exterior G-tetrads were found to be oxidation prone, providing the third line of evidence that the dyes are Type II photooxidants. The present work found that the common dyes fluorescein, rhodamine, BODIPY, or Cy3 can drive G oxidation but with a slow rate and low overall yield. This will likely not impact many experiments using dyes to study nucleic acids except for those that have long exposures with high-intensity lights, such as sequencing-by-synthesis experiments using fluorescence as the readout.

Microbial Community and Biochemical Dynamics of Biological Soil Crusts across a Gradient of Surface Coverage in the Central Mojave Desert.

In this study, we expand upon the biogeography of biological soil crusts (BSCs) and provide molecular insights into the microbial community and biochemical dynamics along the vertical BSC column structure, and across a transect of increasing BSC surface coverage in the central Mojave Desert, CA, United States. Next generation sequencing reveals a bacterial community profile that is distinct among BSCs in the southwestern United States. Distribution of major phyla in the BSC topsoils included Cyanobacteria (33 ± 8%), Proteobacteria (26 ± 6%), and Chloroflexi (12 ± 4%), with Phormidium being the numerically dominant genus. Furthermore, BSC subsurfaces contained Proteobacteria (23 ± 5%), Actinobacteria (20 ± 5%), and Chloroflexi (18 ± 3%), with an unidentified genus from Chloroflexi (AKIW781, order) being numerically dominant. Across the transect, changes in distribution at the phylum (p < 0.0439) and genus (p < 0.006) levels, including multiple biochemical and geochemical trends (p < 0.05), positively correlated with increasing BSC surface coverage. This included increases in (a) Chloroflexi abundance, (b) abundance and diversity of Cyanobacteria, (b) OTU-level diversity in the topsoil, (c) OTU-level differentiation between the topsoil and subsurface, (d) intracellular ATP abundances and catalase activities, and (e) enrichments in clay, silt, and varying elements, including S, Mn, Co, As, and Pb, in the BSC topsoils. In sum, these studies suggest that BSCs from regions of differing surface coverage represent early successional stages, which exhibit increasing bacterial diversity, metabolic activities, and capacity to restructure the soil. Further, these trends suggest that BSC successional maturation and colonization across the transect are inhibited by metals/metalloids such as B, Ca, Ti, Mn, Co, Ni, Mo, and Pb.