Studying Translesion DNA Synthesis Using Xenopus In Vitro Systems.

Cell-free extracts derived from Xenopus eggs have been widely used to decipher molecular pathways involved in several cellular processes including DNA synthesis, the DNA damage response, and genome integrity maintenance. We set out assays using Xenopus cell-free extracts to study translesion DNA synthesis (TLS), a branch of the DNA damage tolerance pathway that allows replication of damaged DNA. Using this system, we were able to recapitulate TLS activities that occur naturally in vivo during early embryogenesis. This chapter describes protocols to detect chromatin-bound TLS factors by western blotting and immunofluorescence microscopy upon induction of DNA damage by UV irradiation, monitor TLS-dependent mutagenesis, and perform proteomic screening.

Computational model of radiation oxygen effect with Monte Carlo simulation: effects of antioxidants and peroxyl radicals.

PURPOSE: Oxygen plays a crucial role in radiation biology. Antioxidants and peroxyl radicals affect the oxygen effect greatly. This study aims to establish a computational model of the oxygen effect and explore the effect attributed to antioxidants and peroxyl radicals. MATERIALS AND METHODS: Oxygen-related reactions are added to our track-structure Monte Carlo code NASIC, including oxygen fixation, chemical repair by antioxidants and damage migration from base-derived peroxyl radicals. Then the code is used to simulate the DNA damage under various oxygen, antioxidant and damage migration rate conditions. The oxygen enhancement ratio(OER) is calculated quantifying by the number of double-strand breaks for each condition. The roles of antioxidants and peroxyl radicals are examined by manipulating the relevant parameters. RESULTS AND CONCLUSIONS: Our results indicate that antioxidants are capable of rapidly restoring DNA radicals through chemical reactions, which compete with natural and oxygen fixation processes. Additionally, antioxidants can react with peroxyl radicals derived from bases, thereby preventing the damage from migrating to DNA strands. By quantitatively accounting for the impact of peroxyl radicals and antioxidants on the OER curves, our study establishes a more precise and comprehensive model of the radiation oxygen effect.

A Practical Site-specific Method for the Detection of Bulky DNA Damages.

Helix-distorting DNA damages block RNA and DNA polymerase, compromising cell function and fate. In human cells, these damages are removed primarily by nucleotide excision repair (NER). Here, we describe damage-sensing PCR (dsPCR), a PCR-based method for the detection of these DNA damages. Exposure to DNA damaging agents results in lower PCR signal in comparison to non-damaged DNA, and repair is measured as the restoration of PCR signal over time. We show that the method successfully detects damages induced by ultraviolet (UV) radiation, by the carcinogenic component of cigarette smoke benzo[a]pyrene diol epoxide (BPDE) and by the chemotherapeutic drug cisplatin. Damage removal measured by dsPCR in a heterochromatic region is less efficient than in a transcribed and accessible region. Furthermore, lower repair is measured in repair-deficient knock-out cells. This straight-forward method could be applied by non-DNA repair experts to study the involvement of their gene-of-interest in repair. Furthermore, this method is fully amenable for high-throughput screening of DNA repair activity.

Ultrafast Formation of a Delocalized Triplet-Excited State in an Epigenetically Modified DNA Duplex under Direct UV Excitation.

Epigenetic modifications impart important functionality to nucleic acids during gene expression but may increase the risk of photoinduced gene mutations. Thus, it is crucial to understand how these modifications affect the photostability of duplex DNA. In this work, the ultrafast formation (<20 ps) of a delocalized triplet charge transfer (CT) state spreading over two stacked neighboring nucleobases after direct UV excitation is demonstrated in a DNA duplex, d(G5fC)9•d(G5fC)9, made of alternating guanine (G) and 5-formylcytosine (5fC) nucleobases. The triplet yield is estimated to be 8 ± 3%, and the lifetime of the triplet CT state is 256 ± 22 ns, indicating that epigenetic modifications dramatically alter the excited state dynamics of duplex DNA and may enhance triplet state-induced photochemistry.

Proximity Estimation and Quantification of Ionizing Radiation-induced DNA Lesions in Aqueous Media using Fluorescence Spectroscopy.

Clustered DNA damage (cluster) or a multiply damaged site, which is a region with two or more lesions within one or two helical turns, has a high mutagenic potential and causes cell death. We quantified fluorophore-labeled lesions and estimated their proximity through fluorescence anisotropy measurements depending on Förster resonance energy transfer (FRET) among the fluorophores close to each other. pUC19 plasmid DNA (2,686 base pairs) dissolved in water or 0.2 M Tris-HCl buffer at a concentration of 10 µg/µL was irradiated by several ionizing radiations with varying linear energy transfers (LET, 0.2-1890 keV/µm). Electrophilic carbonyls (aldehydes and ketones) at abasic sites (APs) produced in DNA were labeled with Alexa Fluor 488 fluorescent dyes with an O-amino functional group. Regardless of the presence or absence of the buffer, AP yields (the number of APs/base pair/Gy) tended to decrease with increasing LET, and the ratio of the AP yield (in 0.2 M Tris-HCl/in water) was less than 0.1 in the LET range of 0.2-200 keV/µm. However, in a higher LET range, the ratios were greater than 0.1. At a low dose, fluorescence anisotropy decreased with increasing LET in 0.2 M Tris-HCl, whereas, in water, this LET dependence was almost insignificant. These findings suggest that 1. the damage distribution on a DNA molecule formed by indirect effects (e.g., by hydroxyl radicals) does not depend on radiation quality and 2. greater LET radiation is more likely to produce a cluster and/or to produce a cluster with shorter distances between lesions by direct effects. This FRET-based proximity estimation of DNA lesions will contribute not only to the identification of clusters and their complexity in a whole genome, but also to the study of their repair mechanism by single-molecular level fluorescence microscopy.

Visualizing the DNA repair process by a photolyase at atomic resolution.

Photolyases, a ubiquitous class of flavoproteins, use blue light to repair DNA photolesions. In this work, we determined the structural mechanism of the photolyase-catalyzed repair of a cyclobutane pyrimidine dimer (CPD) lesion using time-resolved serial femtosecond crystallography (TR-SFX). We obtained 18 snapshots that show time-dependent changes in four reaction loci. We used these results to create a movie that depicts the repair of CPD lesions in the picosecond-to-nanosecond range, followed by the recovery of the enzymatic moieties involved in catalysis, completing the formation of the fully reduced enzyme-product complex at 500 nanoseconds. Finally, back-flip intermediates of the thymine bases to reanneal the DNA were captured at 25 to 200 microseconds. Our data cover the complete molecular mechanism of a photolyase and, importantly, its chemistry and enzymatic catalysis at work across a wide timescale and at atomic resolution.

Filming DNA repair at the atomic level.

Dissection of multistep catalysis by a photoenzyme could inspire green chemistry applications.

DNA as the main target in radiotherapy-a historical overview from first isolation to anti-tumour immune response.

DNA damage is one of the foremost mechanisms of irradiation at the biological level. After the first isolation of DNA by Friedrich Miescher in the 19th century, the structure of DNA was described by Watson and Crick. Several Nobel Prizes have been awarded for DNA-related discoveries. This review aims to describe the historical perspective of DNA in radiation biology. Over the decades, DNA damage has been identified and quantified after irradiation. Depending on the type of sensing, different proteins are involved in sensing DNA damage and repairing the damage, if possible. For double-strand breaks, the main repair mechanisms are non-homologous end joining and homologous recombination. Additional mechanisms are the Fanconi anaemia pathway and base excision repair. Different methods have been developed for the detection of DNA double-strand breaks. Several drugs have been developed that interfere with different DNA repair mechanisms, e.g., PARP inhibitors. These drugs have been established in the standard treatment of different tumour entities and are being applied in several clinical trials in combination with radiotherapy. Over the past decades, it has become apparent that DNA damage mechanisms are also directly linked to the immune response in tumours. For example, cytosolic DNA fragments activate the innate immune system via the cGAS STING pathway.

Modeling of ionizing radiation-induced chromosome aberration and tumor prevalence based on two classes of DNA double-strand breaks clustering in chromatin domains.

There has been some controversy over the use of radiobiological models when modeling the dose-response curves of ionizing radiation (IR)-induced chromosome aberration and tumor prevalence, as those curves usually show obvious non-targeted effects (NTEs) at low doses of high linear energy transfer (LET) radiation. The lack of understanding the contribution of NTEs to IR-induced carcinogenesis can lead to distinct deviations of relative biological effectiveness (RBE) estimations of carcinogenic potential, which are widely used in radiation risk assessment and radiation protection. In this work, based on the initial pattern of two classes of IR-induced DNA double-strand breaks (DSBs) clustering in chromatin domains and the subsequent incorrect repair processes, we proposed a novel radiobiological model to describe the dose-response curves of two carcinogenic-related endpoints within the same theoretical framework. The representative experimental data was used to verify the consistency and validity of the present model. The fitting results indicated that, compared with targeted effect (TE) and NTE models, the current model has better fitting ability when dealing with the experimental data of chromosome aberration and tumor prevalence induced by multiple types of IR with different LETs. Notably, the present model without introducing an NTE term was adequate to describe the dose-response curves of IR-induced chromosome aberration and tumor prevalence with NTEs in low-dose regions. Based on the fitting parameters, the LET-dependent RBE values were calculated for three given low doses. Our results showed that the RBE values predicted by the current model gradually decrease with the increase of doses for the endpoints of chromosome aberration and tumor prevalence. In addition, the calculated RBE was also compared with those evaluated from other models. These analyses show that the proposed model can be used as an alternative tool to well describe dose-response curves of multiple carcinogenic-related endpoints and effectively estimate RBE in low-dose regions.

A study of indirect action's impact on simulated neutron-induced DNA damage.

Objective.The risk of radiobiological stochastic effects associated with neutrons is strongly energy dependent. Recent Monte Carlo studies simulating neutron-irradiated nuclear DNA have demonstrated that this energy dependence is correlated with the relative biological effectiveness (RBE) of neutrons to inflict DNA damage clusters that contain difficult-to-repair double-strand breaks. However, these previous investigations were either limited to modeling direct radiation action or considered the effects of both direct and indirect action together without distinguishing between the two. In this study, we aimed to quantify the influence of indirect action in neutron irradiation scenarios and acquire novel estimations of the energy-dependent neutron RBE for inducing DNA damage clusters due to both direct and indirect action.Approach.We explored the role of indirect action in neutron-induced DNA damage by integrating a validated indirect action model into our existing simulation pipeline. Using this pipeline, we performed track-structure simulations of monoenergetic neutron irradiations (1 eV to 10 MeV) in a nuclear DNA model and analyzed the resulting simple and clustered DNA lesions. We repeated the irradiation simulations for 250 keV x-rays that acted as our reference radiation.Main results.Including indirect action significantly increased the occurrence of DNA lesions. We found that indirect action tends to amplify the damage due to direct action by inducing DNA lesions in the vicinity of directly-induced lesions, resulting in additional and larger damage clusters. Our neutron RBE results are qualitatively similar to but lower in magnitude than the established radiation protection factors and the results of previous similar investigations, due to the greater relative impact of indirect action in photon-induced damage than in neutron-induced damage.Significance.Although our model for neutron-induced DNA damage has some important limitations, our findings suggest that the energy-dependent risk of neutron-induced stochastic effects may not be completely modeled alone by the relative potential of neutrons to inflict clustered lesions via direct and indirect action in DNA damage.

Computational modeling and a Geant4-DNA study of the rejoining of direct and indirect DNA damage induced by low energy electrons and carbon ions.

PURPOSE: DNA double-strand breaks (DSBs) created by ionizing radiations are considered as the most detrimental lesion, which could result in the cell death or sterilization. As the empirical evidence gathered from the cellular and molecular radiation biology has demonstrated significant correlations between the initial and lasting levels of DSBs, gaining knowledge into the DSB repair mechanisms proves vital. Much effort has been invested into understanding the mechanisms triggering the repair and processes engaged after irradiation of cells. Given a mechanistic model, we performed - to our knowledge - the first Monte Carlo study of the expected repair kinetics of carbon ions and electrons using on the one hand Geant4-DNA simulations of electrons for benchmarking purposes and on the other hand quantifying the influence of direct and indirect damage. Our objective was to calculate the DSB repair rates using a repair mechanism for G1 and early S phases of the cell cycle in conjunction with simulations of the DNA damage. MATERIALS AND METHODS: Based on Geant4-DNA simulations of DSB damage caused by electrons and carbon ions - using a B-DNA model and a water sphere of 3 µm radius resembling the mean size of human cells - we derived the kinetics of various biochemical repair processes. RESULTS: The overall repair times of carbon ions increased with the DSB complexity. Comparison of the DSB complexity (DSBc) and repair times as a function of carbon-ion energy suggested that the repair time of no specific fraction of DSBs could solely be explained as a function of DSB complexity. CONCLUSION: Analysis of the carbon-ion repair kinetics indicated that, given a fraction of DSBs, decreasing the energy would result in an increase of the repair time. The disagreements of the calculated and experimental repair kinetics for electrons could, among others, be due to larger damage complexity predicted by simulations or created actually by electrons of comparable energies to x-rays. They are also due to the employed repair mechanisms, which introduce no inherent dependence on the radiation type but make direct use of the simulated DSBs.

Induction of DNA strand breaks and oxidative base damages in plasmid DNA by ultra-high dose rate proton irradiation.

PURPOSE: Radiation cancer therapy with ultra-high dose rate (UHDR) exposure, so-called FLASH radiotherapy, appears to reduce normal tissue damage without compromising tumor response to therapy. The aim of this study was to clarify whether a 59.5 MeV proton beam at an UHDR of 48.6 Gy/s could effectively reduce the DNA damage of pBR322 plasmid DNA in solution compared to the conventional dose rate (CONV) of 0.057 Gy/s. MATERIALS AND METHODS: A simple system, consisting of pBR322 plasmid DNA in 1× Tris-EDTA buffer, was initially employed for proton beam exposure. We then used formamidopyrimidine-DNA glycosylase (Fpg) enzymes. which convert oxidative base damages of oxidized purines to DNA strand breaks, to quantify DNA single strand breaks (SSBs) and double strand breaks (DSBs) by agarose gel electrophoresis. RESULTS: Our findings showed that the SSB induction rate (SSB per plasmid DNA/Gy) at UHDR and the induction of Fpg enzyme sensitive sites (ESS) were significantly reduced in UHDR compared to CONV. However, there was no significant difference in DSB induction and non-DSB cluster damages. CONCLUSIONS: UHDR of a 59.5 MeV proton beam could reduce non-clustered, non-DSB damages, such as SSB and sparsely distributed ESS. However, this effect may not be significant in reducing lethal DNA damage that becomes apparent only in acute radiation effects of mammalian cells and in vivo studies.

Enhanced Single-Strand Breaks of a Nucleic Acid by Gold Nanoparticles under X-ray Irradiation.

The hydroxyl radical concentration-dependent yield of single-strand breaks (SSBs), obtained through correction of scavenging and hindrance effects caused by gold nanoparticles (AuNPs), for fluorophore- and quencher-labeled DNA on AuNPs was 10 times that of free DNA based on fluorescence measurements of X-ray-irradiated DNA on AuNPs. By comparing the fluorescence data that revealed the number of SSBs with the results of mass spectrometry measurements that detected the total damage to DNA, we found that SSBs dominated DNA damage for DNA on AuNPs whereas non-SSB damage dominated for free DNA. The yield of RNA SSBs under X-ray irradiation was similar to that of DNA in the presence of AuNPs, whereas free RNA was more resistive to radiation than DNA. These results indicated the enhanced SSBs were likely catalyzed through the conversion from nucleobase damage to SSBs by AuNPs. The outcome of this work impacts materials and environmental science, sensing, nanotechnology, biology, and medicine.

Comet Assay analysis of DNA strand breaks after exposure to the DNA-incorporated Auger Electron Emitter Iodine-125.

PURPOSE: Ionizing radiation causes various types of DNA damage e.g. single strand breaks (SSB) and double strand breaks (DSB), whereby the SSB/DSB ratio is shifted toward the DSB with increasing LET. For the DNA-incorporated Auger electron emitter Iodine-125 a SSB/DSB ratio of 5.4:1 is calculated based on computer simulations. In the presented work the SSB/DSB ratio of DNA-incorporated Iodine-125 was experimentally determined and compared to external homogenous γ-irradiation. MATERIALS AND METHODS: Iodine-125-iododeoxyuridine (I-125-UdR) was incorporated into the DNA of SCL-II cells and cells were subsequently frozen for decay accumulation. Accordingly, external γ-irradiation (Cs-137) experiments were performed in frozen cells. After exposure the neutral or alkaline Comet Assay was performed to quantify DSB or DSB and SSB, respectively. Automated quantification of the comets was performed using the Olive Tail Moment (Metafer CometScan; MetaSystems). Calculation of absorbed dose for Auger electrons on cellular level is extremely biased due to the exclusive DNA localization of I-125-UdR. To avoid dose calculation the γ-H2AX assay was used in order to allow the comparison of the Comet Assay data between both investigated radiation qualities. RESULTS: For low-LET γ-radiation, a SSB/DSB ratio of 10:1 was determined. In contrast, a lower SSB/DSB ratio of 6:1 was induced by DNA-incorporated Iodine-125 which compares very well to the calculated values of Pomplun and co-authors. CONCLUSION: DNA-incorporated Iodine-125 induces a high-LET type DNA damage pattern in respect to SSB/DSB ratio.

DNA damage by radiation as a function of electron energy and interaction at the atomic level with Monte Carlo simulation.

In radiotherapy, X-ray or heavy ion beams target tumors to cause damage to their cell DNA. This damage is mainly induced by secondary low energy electrons. In this paper, we report the DNA molecular breaks at the atomic level as a function of electron energy and types of electron interactions using of Monte Carlo simulation. The number of DNA single and double strand breaks are compared to those from experimental results based on electron energies. In recent years, DNA atomistic models were introduced but still the simulations consider energy deposition in volumes of DNA or water equivalent material. We simulated a model of atomistic B-DNA in vacuum, forming 1122 base pairs of 30 nm in length. Each atom has been represented by a sphere whose radius equals the radius of van der Waals. We repeatedly simulated 10 million electrons for each energy from 4 eV to 500 eV and counted each interaction type with its position x, y, z in the volume of DNA. Based on the number and types of interactions at the atomic level, the number of DNA single and double strand breaks were calculated. We found that the dissociative electron attachment has the dominant effect on DNA strand breaks at energies below 10 eV compared to excitation and ionization. In addition, it is straightforward with our simulation to discriminate the strand and base breaks as a function of radiation interaction type and energy. In conclusion, the knowledge of DNA damage at the atomic level helps design direct internal therapeutic agents of cancer treatment.

[Exploration of Indole Compounds as Candidate for Radiation Mitigators].

PURPOSE: In this study, to develop radiomitigators capable of the emergency medical care of patients involved in radiation accidents, we investigated the radiomitigative effects and their underlying mechanisms of indole compounds such as DIM, GRM, and INM. METHODS: The human normal fibroblast cell line, MRC-5 cells were administered 0.1% DMSO or each indole compound at 10 µM within 50-60 minutes after X-irradiated with 0-4 Gy. Next, we evaluated the alteration in the number of alive cells, clonogenic potential, DNA double-strand breaks, DNA damage repair activities, and protein expression related to regulate the oxidative stress response. RESULTS: Our results showed that DIM treatment suppressed radiation-induced decrease in the number of alive cells and clonogenic potential. Then, DIM treatment significantly decreased DNA double-strand breaks and highly increased Nrf2 via increased phospho-GSK-3ß (Ser9) expression. These findings suggest that, in part, increased expression of p-GSK-3ß (Ser9) by DIM treatment reduced DNA double-strand breaks via activation of Nrf2, resulting mitigated radiation-induced a decrease in the number of alive cells and clonogenic potential. CONCLUSION: Therefore, DIM, not GRM and INM, is a potential candidate for radiomitigators that can be applied to the radiation emergency medicine.

RADIATION DAMAGE TO DNA PLASMIDS IN THE PRESENCE OF BOROCAPTATES.

Boron derivatives have great potential in cancer diagnostics and treatment. Borocaptates are used in boron neutron capture therapy and potentially in proton boron fusion therapy. This work examines modulation effects of two borocaptate compounds on radiation-induced DNA damage. Aqueous solutions of pBR322 plasmid containing increasing concentrations of borocaptates were irradiated with 60Co gamma rays or 30 MeV protons. Induction of single and double DNA strand breaks was investigated using agarose gel electrophoresis. In this model system, representing DNA without the intervention of cellular repair mechanisms, the boron derivatives acted as antioxidants. Clinically relevant boron concentrations of 40 ppm reduced the DNA single strand breakage seven-fold. Possible mechanisms of the observed effect are discussed.

Nanoscale Calculation of Proton-Induced DNA Damage Using a Chromatin Geometry Model with Geant4-DNA.

Monte Carlo simulations can quantify various types of DNA damage to evaluate the biological effects of ionizing radiation at the nanometer scale. This work presents a study simulating the DNA target response after proton irradiation. A chromatin fiber model and new physics constructors with the ELastic Scattering of Electrons and Positrons by neutral Atoms (ELSEPA) model were used to describe the DNA geometry and the physical stage of water radiolysis with the Geant4-DNA toolkit, respectively. Three key parameters (the energy threshold model for strand breaks, the physics model and the maximum distance to distinguish DSB clusters) of scoring DNA damage were studied to investigate the impact on the uncertainties of DNA damage. On the basis of comparison of our results with experimental data and published findings, we were able to accurately predict the yield of various types of DNA damage. Our results indicated that the difference in physics constructor can cause up to 56.4% in the DNA double-strand break (DSB) yields. The DSB yields were quite sensitive to the energy threshold for strand breaks (SB) and the maximum distance to classify the DSB clusters, which were even more than 100 times and four times than the default configurations, respectively.

Repair of α-particle-induced DNA damage in peripheral blood mononuclear cells after internal ex vivo irradiation with 223Ra.

PURPOSE: As α-emitters for radiopharmaceutical therapies are administered systemically by intravenous injection, blood will be irradiated by α-particles that induce clustered DNA double-strand breaks (DSBs). Here, we investigated the induction and repair of DSB damage in peripheral blood mononuclear cells (PBMCs) as a function of the absorbed dose to the blood following internal ex vivo irradiation with [223Ra]RaCl2. METHODS: Blood samples of ten volunteers were irradiated by adding [223Ra]RaCl2 solution with different activity concentrations resulting in absorbed doses to the blood of 3 mGy, 25 mGy, 50 mGy and 100 mGy. PBMCs were isolated, divided in three parts and either fixed directly (d-samples) or after 4 h or 24 h culture. After immunostaining, the induced γ-H2AX α-tracks were counted. The time-dependent decrease in α-track frequency was described with a model assuming a repair rate R and a fraction of non-repairable damage Q. RESULTS: For 25 mGy, 50 mGy and 100 mGy, the numbers of α-tracks were significantly increased compared to baseline at all time points. Compared to the corresponding d-samples, the α-track frequency decreased significantly after 4 h and after 24 h. The repair rates R were (0.24 ± 0.05) h-1 for 25 mGy, (0.16 ± 0.04) h-1 for 50 mGy and (0.13 ± 0.02) h-1 for 100 mGy, suggesting faster repair at lower absorbed doses, while Q-values were similar. CONCLUSION: The results obtained suggest that induction and repair of the DSB damage depend on the absorbed dose to the blood. Repair rates were similar to what has been observed for irradiation with low linear energy transfer.

Never Cared for What They Do: High Structural Stability of Guanine-Quadruplexes in the Presence of Strand-Break Damage.

DNA integrity is an important factor that assures genome stability and, more generally, the viability of cells and organisms. In the presence of DNA damage, the normal cell cycle is perturbed when cells activate their repair processes. Although efficient, the repair system is not always able to ensure complete restoration of gene integrity. In these cases, mutations not only may occur, but the accumulation of lesions can either lead to carcinogenesis or reach a threshold that induces apoptosis and programmed cell death. Among the different types of DNA lesions, strand breaks produced by ionizing radiation are the most toxic due to the inherent difficultly of repair, which may lead to genomic instability. In this article we show, by using classical molecular simulation techniques, that compared to canonical double-helical B-DNA, guanine-quadruplex (G4) arrangements show remarkable structural stability, even in the presence of two strand breaks. Since G4-DNA is recognized for its regulatory roles in cell senescence and gene expression, including oncogenes, this stability may be related to an evolutionary cellular response aimed at minimizing the effects of ionizing radiation.