Cellular respiration and ionizing radiation generate 5’,8-cyclo-2’-deoxyribonucleosides, an especial type of DNA damage that involves two modifications in the same nucleotide. These lesions evade the action of base excision glycosylases and their removal is a function of the nucleotide excision repair pathway. Diastereomeric 5’,8-cyclo-2’-deoxyadenosine block mammalian DNA replication, diminish the levels of DNA transcription and induce transcriptional mutagenesis. Using solution state NMR spectroscopy and restrained molecular dynamics simulations, we have determined the structure of an undecameric DNA duplex having a centrally located (5’S)-5’,8-cyclo-2’-deoxyadenosine residue paired to T. The damaged duplex structure is a right-handed helix having Watson-Crick base-pair alignments throughout and 2-deoxyribose puckers within the B-form conformation. Only small structural perturbations are observed at the lesion-containing and 5’-flanking base-pair. The 2-deoxyribose of the damaged nucleotide adopts the O4’-exo conformation and the S-cdA•T base-pair is propeller twisted. The 5’-lesion-flanking base is tilted forming a significantly buckled base-pair with its partner guanine. Analysis of UV-melting curves indicates mild thermal and thermodynamic destabilization on the damaged duplex. The S-cdA•T duplex structure shows many similarities and some intriguing differences with the recently reported structure of an S-cdG•dC duplex31 that suggest different lesion site dynamics.
The near exponential proliferation of published Raman microspectroscopic applications over the last decade bears witness to the strengths and versatility of this technology. However, laser-induced fluorescence often severely impedes its application to biological samples. Here we report a new approach for near complete elimination of laser-induced background fluorescence in highly pigmented biological specimens (e.g., microalgae) enabling interrogation by Raman microspectroscopy. Our simple chemiphotobleaching method combines mild hydrogen peroxide oxidation with broad spectrum visible light irradiation of the entire specimen. This treatment permits observing intracellular distributions of macromolecular pools, isotopic tracers, and even viral propagation within cells previously not amenable to Raman microspectroscopic examination. Our approach demonstrates the potential for confocal Raman microspectroscopy becoming an indispensable tool to obtain spatially-resolved data on the chemical composition of highly fluorescent biological samples from individual cells to environmental samples.
The addition of hydroxyl radicals to the C8 position of guanine can lead to the formation of 2,6-diamino-4-hydroxy-5-formamido-2′-deoxypyrimidine (Fapy-dG) lesion, whose endogenous levels in cellular DNA rival those of 8-oxo-7,8-dihydroxy-2′-deoxyguanosine. Despite its prevalence, the structure of duplex DNA containing Fapy-dG is unknown. We have prepared an undecameric duplex containing a centrally located β-cFapy-dG residue paired to dC and determined its solution structure by high resolution NMR spectroscopy and restrained molecular dynamic simulations. The damaged duplex adopts a right-handed helical structure all residues in an anti conformation, forming Watson-Crick base pair alignments, and 2-deoxyribose conformations in the C2′-endo/C1′-exo range. The formamido group of Fapy rotates out of the pyrimidine plane and is present on the Z and E configurations that equilibrate with an approximate 2:1 population ratio. The two isomeric duplexes show similar lesion induced deviations from a canonical B-from DNA conformation that are minor and limited to the central three-base-pair segment of the duplex, affecting the stacking interactions with the 5-lesion-neighboring residue. We discuss the implications of our observations for translesion synthesis during DNA replication and the recognition of Fapy-dG by DNA glycosylases.
A new method to measure growth rates of individual photoautotrophic cells by combining stable isotope probing (SIP) and single-cell resonance Raman microspectrometry is introduced. This report explores optimal experimental design and the theoretical underpinnings for quantitative responses of Raman spectra to cellular isotopic composition. Resonance Raman spectra of isogenic cultures of the cyanobacterium, Synechococcus sp., grown in 13C-bicarbonate revealed linear covariance between wavenumber (cm−1) shifts in dominant carotenoid Raman peaks and a broad range of cellular 13C fractional isotopic abundance. Single-cell growth rates were calculated from spectra-derived isotopic content and empirical relationships. Growth rates among any 25 cells in a sample varied considerably; mean coefficient of variation, CV, was 29 ± 3% (σ/falsemml-overlinex¯), of which only ~2% was propagated analytical error. Instantaneous population growth rates measured independently by in vivo fluorescence also varied daily (CV ≈ 53%) and were statistically indistinguishable from single-cell growth rates at all but the lowest levels of cell labeling. SCRR censuses of mixtures prepared from Synechococcus sp. and T. pseudonana (a diatom) populations with varying 13C-content and growth rates closely approximated predicted spectral responses and fractional labeling of cells added to the sample. This approach enables direct microspectrometric interrogation of isotopically- and phylogenetically-labeled cells and detects as little as 3% changes in cellular fractional labeling. This is the first description of a non-destructive technique to measure single-cell photoautotrophic growth rates based on Raman spectroscopy and well-constrained assumptions, while requiring few ancillary measurements.
The suitability of stable isotope probing (SIP) and Raman microspectroscopy to measure growth rates of heterotrophic bacteria at the single-cell level was evaluated. Label assimilation into E. coli biomass during growth on a complex 13 C-labeled carbon source was monitored in time course experiments. 13 C-incorporation into various biomolecules was measured by spectral “red shifts” of Raman-scattered emissions. The 13 C- and 12 C-isotopologues of the amino acid phenylalanine (Phe) proved to be a quantitatively accurate reporter molecules of cellular isotopic fractional abundances ( f cell ). Values of f cell determined by Raman microspectroscopy and independently by isotope-ratio mass spectrometry (IRMS) over a range of isotopic enrichments were statistically indistinguishable. Progressive labeling of Phe in E. coli cells among a range of 13 C/ 12 C organic substrate admixtures occurred predictably through time. Relative isotopologue abundances of Phe determined by Raman spectral analysis enabled accurate calculation of bacterial growth rates as confirmed independently by optical density (OD) measurements. Results demonstrate that combining stable isotope probing (SIP) and Raman microspectroscopy can be a powerful tool for studying bacterial growth at the single-cell level when grown on defined or complex organic 13 C-carbon sources even in mixed microbial assemblages. Importance: Population growth dynamics and individual cell growth rates are the ultimate expressions of a microorganism’s fitness to its environmental conditions, whether natural or engineered. Natural habitats and many industrial settings harbor complex microbial assemblages. Their heterogeneity in growth responses to existing and changing conditions is often difficult to grasp by standard methodologies. In this proof of concept study, we tested whether Raman microspectroscopy can reliably quantify assimilation of isotopically-labeled nutrients into E. coli cells and enable determination of individual growth rates among heterotrophic bacteria. Raman-derived growth rate estimates were statistically indistinguishable from those derived by standard optical density measurements of the same cultures. Raman microspectroscopy also can be combined with methods for phylogenetic identification. We report development of Raman-based techniques that enable researchers to directly link genetic identity to functional traits and rate measurements of single cells within mixed microbial assemblages, currently a major technical challenge in microbiological research.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.