Global and transcription-coupled repair of 8-oxoG is initiated by nucleotide excision repair proteins.

UV-DDB, consisting of subunits DDB1 and DDB2, recognizes UV-induced photoproducts during global genome nucleotide excision repair (GG-NER). We recently demonstrated a noncanonical role of UV-DDB in stimulating base excision repair (BER) which raised several questions about the timing of UV-DDB arrival at 8-oxoguanine (8-oxoG), and the dependency of UV-DDB on the recruitment of downstream BER and NER proteins. Using two different approaches to introduce 8-oxoG in cells, we show that DDB2 is recruited to 8-oxoG immediately after damage and colocalizes with 8-oxoG glycosylase (OGG1) at sites of repair. 8-oxoG removal and OGG1 recruitment is significantly reduced in the absence of DDB2. NER proteins, XPA and XPC, also accumulate at 8-oxoG. While XPC recruitment is dependent on DDB2, XPA recruitment is DDB2-independent and transcription-coupled. Finally, DDB2 accumulation at 8-oxoG induces local chromatin unfolding. We propose that DDB2-mediated chromatin decompaction facilitates the recruitment of downstream BER proteins to 8-oxoG lesions.

Radicals generated in alternating guanine-cytosine duplexes by direct absorption of low-energy UV radiation.

Recent studies have evidenced that oxidatively damaged DNA, which potentially leads to carcinogenic mutations and aging, may result from the direct absorption of low-energy photons (>250 nm). Herein, the primary species, i.e., ejected electrons and base radicals associated with such damage in duplexes with an alternating guanine-cytosine sequence are quantified by nanosecond transient absorption spectroscopy. The one-photon ionization quantum yield at 266 nm is 1.2 × 10-3, which is similar to those reported previously for adenine-thymine duplexes. This means that the simple presence of guanine, the nucleobase with the lowest ionization potential, does not affect photo-ionization. The transient species detected after 3 µs are identified as deprotonated guanine radicals, which decay with a half-time of 2.5 ms. Spectral assignment is made with the help of quantum chemistry calculations (TD-DFT), which for the first time, provide reference absorption spectra for guanine radicals in duplexes. In addition, our computed spectra predict the changes in transient absorption expected for hole localization as well as deprotonation (to cytosine and bulk water) and hydration of the radical cation.

Mutagenic potential of 8-oxo-7,8-dihydroguanine (8-oxoG) is influenced by nearby clustered lesions.

Ionizing radiation causes various different types of DNA damage. If not repaired, DNA damage can have detrimental effects. Previous studies indicate that the spatial distribution of DNA lesions induced by ionizing radiation is highly relevant to the ensuing biological effects. Clustered DNA damage, consisting of DNA lesions in close proximity, has been studied in detail, and has enhanced mutagenic potential depending on the configuration of the lesions. However, it is not known whether clustered DNA damage affects the mutagenic potential of a sufficiently separated, isolated lesion. Using synthetic damage constructs, we investigated the mutagenic potential of an isolated 8-oxo-7,8-dihydroguanine (8-oxoG) separated by at least 7 bp from other lesions. Under the spatial distribution of DNA lesions tested in the present study, neighboring clustered DNA lesions likely retarded the processing of the isolated 8-oxoG and resulted in enhanced mutation frequency. However, the enhanced mutagenic potential was dependent on which strand the isolated 8-oxoG was located. Our results indicate that the processing of a bi-stranded cluster could affect the mutagenic outcome of a nearby isolated lesion, separated up to ∼20 bp.

Optochemical control of gene expression by photocaged guanine and riboswitches.

Optical control of biomolecules via engineered proteins or photoactive small molecules has had a profound impact on biology. However, optochemical tools to control RNA functions in living cells are relatively limited. We synthesized a photoactivatable (photocaged) guanine to modulate gene expression under riboswitch control in both mammalian cells and Escherichia coli by light.

Role of photoresponse of π electrons in light-driven DNA dissociations.

The role of photoresponse of π electrons in light-driven DNA dissociations is theoretically studied. A new model combining the Peyrard-Bishop-Dauxois model and the charge ladder model is first proposed. Then the evolutions of π-electronic states and H-bond stretching in the light-driven DNA dissociations are studied. The results show that light irradiation will induce ultrafast charge redistribution among bases, leading to the precursory insulator-to-metallic transition. This electronic transition will assist DNA to dissociate. Effects of screened Coulomb interactions on dissociation dynamics is emphatically discussed. Finally, it is also found that light-driven DNA dissociation preferentially occurs in the adenine-thymine-rich region rather than the guanine-cytosine-rich region.

Photoionization spectroscopy of nucleobases and analogues in the gas phase using synchrotron radiation as excitation light source.

We review here the photoionization and photoelectron spectroscopy of the gas phase nucleic acid bases adenine, thymine, uracil, cytosine, and guanine, as well as the three base analogues 2-hydroxyisoquinoline, 2-pyridone, and δ-valerolactam in the vacuum ultraviolet (VUV) spectral regime. The chapter focuses on experimental work performed with VUV synchrotron radiation and related ab initio quantum chemical calculations of higher excited states beyond the ionization energy. After a general part, where experimental and theoretical techniques are described in detail, key results are presented by order of growing complexity in the spectra of the molecules. Here we concentrate on (1) the accurate determination of ionization energies of isolated gas phase NABs and investigation of the vibrational structure of involved ionic states, including their mutual vibronic couplings, (2) the treatment of tautomerism after photoionization, in competition with other intramolecular processes, (3) the study of fragmentation of these molecular systems at low and high internal energies, and (4) the study of the evolution of the covalent character of hydrogen bonding upon substitution, i.e., examination of electronic effects (acceptor, donor, etc.).

Photochemistry of nucleic acid bases and their thio- and aza-analogues in solution.

The steady-state and time-resolved photochemistry of the natural nucleic acid bases and their sulfur- and nitrogen-substituted analogues in solution is reviewed. Emphasis is given to the experimental studies performed over the last 3-5 years that showcase topical areas of scientific inquiry and those that require further scrutiny. Significant progress has been made toward mapping the radiative and nonradiative decay pathways of nucleic acid bases. There is a consensus that ultrafast internal conversion to the ground state is the primary relaxation pathway in the nucleic acid bases, whereas the mechanism of this relaxation and the level of participation of the (1)πσ*, (1) nπ*, and (3)ππ* states are still matters of debate. Although impressive research has been performed in recent years, the microscopic mechanism(s) by which the nucleic acid bases dissipate excess vibrational energy to their environment, and the role of the N-glycosidic group in this and in other nonradiative decay pathways, are still poorly understood. The simple replacement of a single atom in a nucleobase with a sulfur or nitrogen atom severely restricts access to the conical intersections responsible for the intrinsic internal conversion pathways to the ground state in the nucleic acid bases. It also enhances access to ultrafast and efficient inter-system crossing pathways that populate the triplet manifold in yields close to unity. Determining the coupled nuclear and electronic pathways responsible for the significantly different photochemistry in these nucleic acid base analogues serves as a convenient platform to examine the current state of knowledge regarding the photodynamic properties of the DNA and RNA bases from both experimental and computational perspectives. Further investigations should also aid in forecasting the prospective use of sulfur- and nitrogen-substituted base analogues in photochemotherapeutic applications.

Excited states in DNA strands investigated by ultrafast laser spectroscopy.

Ultrafast laser experiments on carefully selected DNA model compounds probe the effects of base stacking, base pairing, and structural disorder on excited electronic states formed by UV absorption in single and double DNA strands. Direct π-orbital overlap between two stacked bases in a dinucleotide or in a longer single strand creates new excited states that decay orders of magnitude more slowly than the generally subpicosecond excited states of monomeric bases. Half or more of all excited states in single strands decay in this manner. Ultrafast mid-IR transient absorption experiments reveal that the long-lived excited states in a number of model compounds are charge transfer states formed by interbase electron transfer, which subsequently decay by charge recombination. The lifetimes of the charge transfer states are surprisingly independent of how the stacked bases are oriented, but disruption of π-stacking, either by elevating temperature or by adding a denaturing co-solvent, completely eliminates this decay channel. Time-resolved emission measurements support the conclusion that these states are populated very rapidly from initial excitons. These experiments also reveal the existence of populations of emissive excited states that decay on the nanosecond time scale. The quantum yield of these states is very small for UVB/UVC excitation, but increases at UVA wavelengths. In double strands, hydrogen bonding between bases perturbs, but does not quench, the long-lived excited states. Kinetic isotope effects on the excited-state dynamics suggest that intrastrand electron transfer may couple to interstrand proton transfer. By revealing how structure and non-covalent interactions affect excited-state dynamics, on-going experimental and theoretical studies of excited states in DNA strands can advance understanding of fundamental photophysics in other nanoscale systems.

UV-excitation from an experimental perspective: frequency resolved.

Electronic spectroscopy of DNA bases in the gas phase provides detailed information about the electronic excitation, which places the molecule in the Franck-Condon region in the excited state and thus prepares the starting conditions for excited-state dynamics. Double resonance or hole-burning spectroscopy in the gas phase can provide such information with isomer specificity, probing the starting potential energy landscape as a function of tautomeric form, isomeric structure, or hydrogen bonded or stacked cluster structure. Action spectroscopy, such REMPI, can be affected by excited-state lifetimes.

Modified nucleobases.

Various molecules which are similar to the natural nucleobases exist in nature or have been synthetically developed. In this chapter we review work on the photophysical properties of several modified nucleobases, focusing particularly on how these properties differ from those of the natural nucleobases. We discuss studies that give physical insight into how the molecular structure can be related to photophysical properties with many of these studies being theoretical. One useful photophysical property is the ability to fluoresce with high quantum yields. Natural bases practically do not fluoresce, so being able to design molecules that fluoresce is a goal of practical importance. Many of the modified nucleobases discussed in this review are fluorescent analogues, analogues that have very different fluorescent properties from the natural bases. The studies reviewed here may provide ways to design other analogues with a set of desired properties.

Electronic excitations in Guanine quadruplexes.

Guanine rich DNA strands, such as those encountered at the extremities of human chromosomes, have the ability to form four-stranded structures (G-quadruplexes) whose building blocks are guanine tetrads. G-quadruplex structures are intensively studied in respect of their biological role, as targets for anticancer therapy and, more recently, of their potential applications in the field of molecular electronics. Here we focus on their electronic excited states which are compared to those of non-interacting mono-nucleotides and those of single and double stranded structures. Particular emphasis is given to excited state relaxation processes studied by time-resolved fluorescence spectroscopy from femtosecond to nanosecond time scales. They include ultrafast energy transfer and trapping of ππ* excitations by charge transfer states. The effect of various structural parameters, such as the nature of the metal cations located in the central cavity of G-quadruplexes, the number of tetrads or the conformation of the constitutive single strands, are examined.

Excited states behavior of nucleobases in solution: insights from computational studies.

We review the most significant results obtained in the study of isolated nucleobases in solution by quantum mechanical methods, trying to highlight also the most relevant open issues. We concisely discuss some methodological issues relevant to the study of molecular electronic excited molecular states in condensed phases, focussing on the methods most commonly applied to the study of nucleobases, i.e. continuum models as the Polarizable Continuum Model and explicit solvation models. We analyse how the solvent changes the relative energy of the lowest energy excited states in the Franck-Condon region, their minima and the Conical Intersections among the different states, interpreting the experimental optical spectra, both steady state and time-resolved. Several methods are available for accurately including solvent effects in the Franck-Condon region, and for most of the nucleobases the solvent shift on the different excited states can be considered assessed. The study of the excited state decay, both radiative and non-radiative, in solution still poses instead significant theoretical challenges.

Computational modeling of photoexcitation in DNA single and double strands.

The photoexcitation of DNA strands triggers extremely complex photoinduced processes, which cannot be understood solely on the basis of the behavior of the nucleobase building blocks. Decisive factors in DNA oligomers and polymers include collective electronic effects, excitonic coupling, hydrogen-bonding interactions, local steric hindrance, charge transfer, and environmental and solvent effects. This chapter surveys recent theoretical and computational efforts to model real-world excited-state DNA strands using a variety of established and emerging theoretical methods. One central issue is the role of localized vs delocalized excitations and the extent to which they determine the nature and the temporal evolution of the initial photoexcitation in DNA strands.

Excitation of nucleobases from a computational perspective II: dynamics.

This chapter is devoted to unravel the relaxation processes taking place after photoexcitation of isolated DNA/RNA nucleobases in gas phase from a time-dependent perspective. To this aim, several methods are at hand, ranging from full quantum dynamics to various flavours of semiclassical or ab initio molecular dynamics, each with its advantages and its limitations. As this contribution shows, the most common approach employed up to date to learn about the deactivation of nucleobases in gas phase is a combination of the Tully surface hopping algorithm with on-the-fly CASSCF calculations. Different dynamics methods or, even more dramatically, different electronic structure methods can provide different dynamics. A comprehensive review of the different mechanisms suggested for each nucleobase is provided and compared to available experimental time scales. The results are discussed in a general context involving the effects of the different applied electronic structure and dynamics methods. Mechanistic similarities and differences between the two groups of nucleobases - the purine derivatives (adenine and guanine) and the pyrimidine derivatives (thymine, uracil, and cytosine) - are elucidated. Finally, a perspective on the future of dynamics simulations in the context of nucleobase relaxation is given.

Excitation of nucleobases from a computational perspective I: reaction paths.

The main intrinsic photochemical events in nucleobases can be described on theoretical grounds within the realm of non-adiabatic computational photochemistry. From a static standpoint, the photochemical reaction path approach (PRPA), through the computation of the respective minimum energy path (MEP), can be regarded as the most suitable strategy in order to explore the electronically excited isolated nucleobases. Unfortunately, the PRPA does not appear widely in the studies reported in the last decade. The main ultrafast decay observed experimentally for the gas-phase excited nucleobases is related to the computed barrierless MEPs from the bright excited state connecting the initial Franck-Condon region and a conical intersection involving the ground state. At the highest level of theory currently available (CASPT2//CASPT2), the lowest excited (1)(ππ*) hypersurface for cytosine has a shallow minimum along the MEP deactivation pathway. In any case, the internal conversion processes in all the natural nucleobases are attained by means of interstate crossings, a self-protection mechanism that prevents the occurrence of photoinduced damage of nucleobases by ultraviolet radiation. Many alternative and secondary paths have been proposed in the literature, which ultimately provide a rich and constructive interplay between experimentally and theoretically oriented research.

Increased mutability and decreased repairability of a three-lesion clustered DNA-damaged site comprised of an AP site and bi-stranded 8-oxoG lesions.

PURPOSE: Ionizing radiation induces DNA damage, some of which are present in clusters, defined as two or more lesions within one to two helical turns of DNA by passage of a single radiation track. These clusters are thought to contribute to the detrimental effects of radiation, in part due to the compromised repair of clustered DNA damaged sites. MATERIALS AND METHODS: The repair of three-lesion cluster present in oligonucleotides were determined in vitro using the hamster cell line CHO-K1 nuclear extract or purified proteins involved in base excision repair. The mutagenic potential of these clusters present in a plasmid was determined using an Escherichia coli reporter assay. RESULTS: We have shown that the repair of an abasic (AP) site within a three-lesion cluster, comprised of an AP site and bi-stranded 8-oxo-7,8-dihydroguanine (8-oxoG) lesions, is retarded compared to that of an isolated AP site in an in vitro base excision repair (BER) assay. Further, the mutation frequency of the clustered damaged site is up to three times greater than that of an isolated 8-oxoG lesion. CONCLUSIONS: As a consequence of enhanced mutagenic potential of clusters, non-double-strand break (DSB) DNA damage may contribute to the detrimental effects of radiation, in addition to the effects of DSB.

Atomic structure from large-area, low-dose exposures of materials: a new route to circumvent radiation damage.

Beam-induced structural modifications are a major nuisance in the study of materials by high-resolution electron microscopy. Here, we introduce a new approach to circumvent the radiation damage problem by a statistical treatment of large, noisy, low-dose data sets of non-periodic configurations (e.g. defects) in the material. We distribute the dose over a mixture of different defect structures at random positions and with random orientations, and recover representative model images via a maximum likelihood search. We demonstrate reconstructions from simulated images at such low doses that the location of individual entities is not possible. The approach may open a route to study currently inaccessible beam-sensitive configurations.

Radiopacity evaluation of calcium aluminate cement containing different radiopacifying agents.

INTRODUCTION: The aim of this study was to evaluate the radiopacity of calcium aluminate cement (EndoBinder) with 3 different radiopacifiers (bismuth oxide, zinc oxide, or zirconium oxide) in comparison with gray mineral trioxide aggregate (GMTA), white MTA, and dental structures (enamel and dentin). METHODS: Eighteen test specimens of each cement with thicknesses of 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mm (n = 3) were made by using a stainless steel matrix and were adapted to a standardizing device (8 × 7 cm) with a graduated aluminum stepwedge varying from 2.0-16.0 mm in thickness. To compare the radiopacity of the cements with that of dental structures, slices of first molars with a thickness increasing from 0.5-3.0 mm were obtained and placed on the standardizing device. One occlusal radiograph for each tested cement was taken, with exposure time of 0.1 seconds and focus-film distance of 20 cm. Films were processed in an automatic device, and the mean radiopacity values were obtained by using a photodensitometer. RESULTS: Mean values showed that the thicker the specimen was, the greater was its radiopacity. Only EndoBinder + bismuth oxide (EBBO) and GMTA demonstrated radiopacity values greater than 3.0 mm of the aluminum scale for all thicknesses. When zinc oxide was used as radiopacifier agent, EndoBinder only reached the desired radiopacity with a thickness of 2.0 mm, and with zirconium oxide it was 2.5 mm. CONCLUSIONS: Bismuth oxide was the most efficient radiopacifier for EndoBinder, providing adequate radiopacity in all studied thicknesses, as recommended by ISO 6876, being similar to GMTA.

The sunscreen agent 2-phenylbenzimidazole-5-sulfonic acid photosensitizes the formation of oxidized guanines in cellulo after UV-A or UV-B exposure.

The sunscreen agent 2-phenylbenzimidazole-5-sulfonic acid (PBSA) is water soluble and is widely used in the cosmetic industry because it absorbs strongly at UV-B wavelengths. Previous studies have shown that PBSA, photoexcited by UV-B, oxidizes guanine bases in vitro. Because of its potential phototoxic effect, it is important to determine whether PBSA photosensitizes in cellulo the formation of oxidatively generated DNA damage on UV exposure. For this purpose, we investigated, in vitro and in cellulo, the effect of PBSA on DNA bases after UV-A or UV-B irradiation. To monitor the formation of oxidized bases and cyclobutane pyrimidine dimers (CPDs), DNA was digested either with FaPy-DNA glycosylase and endonuclease III or with T4 endonuclease V and photolyase, then analyzed by means of neutral- and glyoxal-agarose gel electrophoresis and ligation-mediated PCR. In cellulo, we found that PBSA provided good protection against CPD formation after UV-B exposure. However, PBSA also photosensitized oxidized guanines on UV-A and UV-B irradiation. Our results indicate that PBSA has the potential to function as a double-edged sword toward DNA and question its suitability for sunscreen applications.

Impact of ketone and amino on the inner shell of guanine.

Removal of the functional groups of guanine, i.e. ketone and amino, one by one produces model molecules of hypoxanthine, 2-aminopurine and unsubstituted purine. The impact of the ketone and amino moieties on guanine is revealed using their atomic-site-based inner-shell electronic properties and spectra. A density functional theory based model has been employed to study the model molecules. Electronic properties, such as Hirshfeld charges and inner-shell chemical shift, are found to be both site-dependent and moiety-dependent. The site-based inner-shell chemical shift of the species exhibits a simple linear correlation, although certain similarities among the model molecules regroup the species into two pairs of purine and 2-aminopurine, as well as hypoxanthine and guanine.