The Arabidopsis ATR-SOG1 signaling module regulates pleiotropic developmental adjustments in response to 3'-blocked DNA repair intermediates.

Base excision repair and active DNA demethylation produce repair intermediates with DNA molecules blocked at the 3'-OH end by an aldehyde or phosphate group. However, both the physiological consequences of these accumulated single-strand DNAs break with 3'-blocked ends (DNA 3'-blocks) and the signaling pathways responding to unrepaired DNA 3'-blocks remain unclear in plants. Here, we investigated the effects of DNA 3'-blocks on plant development using the zinc finger DNA 3'-phosphoesterase (zdp) AP endonuclease2 (ape2) double mutant, in which 3'-blocking residues are poorly repaired. The accumulation of DNA 3'-blocked triggered diverse developmental defects that were dependent on the ATM and RAD3-related (ATR)-suppressor of gamma response 1 (SOG1) signaling module. SOG1 mutation rescued the developmental defects of zdp ape2 leaves by preventing cell endoreplication and promoting cell proliferation. However, SOG1 mutation caused intensive meristematic cell death in the radicle of zdp ape2 following germination, resulting in rapid termination of radicle growth. Notably, mutating FORMAMIDOPYRIMIDINE DNA GLYCOSYLASE (FPG) in zdp ape2 sog1 partially recovered its radicle growth, demonstrating that DNA 3'-blocks generated by FPG caused the meristematic defects. Surprisingly, despite lacking a functional radicle, zdp ape2 sog1 mutants compensated the lack of root growth by generating anchor roots having low levels of DNA damage response. Our results reveal dual roles of SOG1 in regulating root establishment when seeds germinate with excess DNA 3'-blocks.

Repair-driven DNA tetrahedral nanomachine combined with DNAzyme for 8-oxo guanine DNA glycosylase activity assay, drug screening and intracellular imaging.

8-oxo guanine DNA glycosylase (8-oxoG DNA glycosylase), a crucial DNA repair enzyme, is essential for maintaining genome integrity and preventing diseases caused by DNA oxidative damage. Imaging 8-oxoG DNA glycosylase in living cells requires a dependable technique. In this study, we designed a DNAzyme-modified DNA tetrahedral nanomachine (DTDN) powered by 8-oxoG restoration. Incorporating a molecular beacon probe (MB), the constructed platform was used for amplified in situ monitoring of 8-oxoG DNA glycosylase. Under normal conditions, duplexing with a complementary strand modified with two 8-oxoG sites inhibited the activity of DNAzyme. The restoration of DNAzyme activity by the repair of intracellular 8-oxoG DNA glycosylase on 8-oxoG bases can initiate a signal amplification reaction. This detection system can detect 8-oxoG DNA glycosylase activity linearly between 0 and 20 U mL-1, with a detection limit as low as 0.52 U mL-1. Using this method, we were able to screen 14 natural compounds and identify 6 of them as 8-oxoG DNA glycosylase inhibitors. In addition, a novel approach was utilized to assess the activity of 8-oxoG DNA glycosylase in living cells. In conclusion, this method provides a universal tool for monitoring the activity of 8-oxoG DNA glycosylase in vitro and in living cells, which holds great promise for elucidating the enzyme's functionality and facilitating drug screening endeavors.

Structural and functional determinants of the archaeal 8-oxoguanine-DNA glycosylase AGOG for DNA damage recognition and processing.

8-Oxoguanine (GO) is a major purine oxidation product in DNA. Because of its highly mutagenic properties, GO absolutely must be eliminated from DNA. To do this, aerobic and anaerobic organisms from the three kingdoms of life have evolved repair mechanisms to prevent its deleterious effect on genetic integrity. The major way to remove GO is the base excision repair pathway, usually initiated by a GO-DNA glycosylase. First identified in bacteria (Fpg) and eukaryotes (OGG1), GO-DNA glycosylases were more recently identified in archaea (OGG2 and AGOG). AGOG is the less documented enzyme and its mode of damage recognition and removing remains to be clarified at the molecular and atomic levels. This study presents a complete structural characterisation of apo AGOGs from Pyrococcus abyssi (Pab) and Thermococcus gammatolerans (Tga) and the first structure of Pab-AGOG bound to lesion-containing single- or double-stranded DNA. By combining X-ray structure analysis, site directed mutagenesis and biochemistry experiments, we identified key amino acid residues of AGOGs responsible for the specific recognition of the lesion and the base opposite the lesion and for catalysis. Moreover, a unique binding mode of GO, involving double base flipping, never observed for any other DNA glycosylases, is revealed. In addition to unravelling the properties of AGOGs, our study, through comparative biochemical and structural analysis, offers new insights into the evolutionary plasticity of DNA glycosylases across all three kingdoms of life.

Assay conditions for estimating differences in base excision repair activity with Fpg-modified comet assay.

DNA repair is an essential agent in cancer development, progression, prognosis, and response to therapy. We have adapted a cellular repair assay based on the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay to assess DNA repair kinetics. The removal of oxidized nucleobases over time (0-480 min) was analyzed in peripheral blood mononuclear cells (PBMCs) and 8 cell lines. DNA damage was induced by exposure to either Ro19-8022 plus visible light or potassium bromate (KBrO3). The initial amount of damage induced by Ro 19-8022 plus light varied between cell lines, and this was apparently associated with the rate of repair. However, the amount of DNA damage induced by KBrO3 varied less between cell types, so we used this agent to study the kinetics of DNA repair. We found an early phase of ca. 60 min with fast removal of Fpg-sensitive sites, followed by slower removal over the following 7 h. In conclusion, adjusting the initial damage at T0 to an equal level can be achieved by the use of KBrO3, which allows for accurate analysis of subsequent cellular DNA repair kinetics in the first hour after exposure.

Structure of the major oxidative damage 7,8-dihydro-8-oxoguanine presented into a catalytically competent DNA glycosylase.

If left unrepaired, the major oxidative DNA lesion 7,8-dihydro-8-oxoguanine (oxoG) promotes G-to-T transversions by favorably adopting a syn conformation and base pairing with dATP during replication. The human oxoG DNA glycosylase hOGG1 senses and removes oxoG amid millions-fold excess of guanine, thereby counteracting the genotoxic effects of the major oxidative damage. Crystal structures of hOGG1 in complex with oxoG-containing DNA have provided key insights into the lesion recognition and catalysis mechanisms of the enzyme. These lesion-recognition complex (LRC) structures typically involve a catalytically inactive hOGG1 mutant, where one of the catalytic-site amino acid residues is mutated to prevent the cleavage of oxoG. The use of a catalytically incompetent hOGG1 mutant has thus precluded understanding of unscathed interactions between oxoG and hOGG1 catalytic site as well as interactions among catalytic-site amino acid residues. As an orthogonal approach to visualize such interactions, we have co-crystallized a catalytically competent hOGG1 bound to 2'-fluoro-oxodG-containing DNA, a transition state destabilizing inhibitor that binds hOGG1 but is not processed by the enzyme. In this fluorinated lesion-recognition complex (FLRC), the 8-oxo moiety of oxoG is recognized by Gly42 and the Watson-Crick edge of oxoG is contacted by Gln315 and Pro266. The previously observed salt bridge between Lys249 and Cys253 is lacking in the FLRC, suggesting Lys249 is primed by Cys253 and poised for nucleophilic attack on C1' of oxodG. Overall, hOGG1 FLRC marks the first structure of oxoG presented into an intact catalytic site of hOGG1 and provides complementary insights into the glycosylase mechanisms of the enzyme.

Profiling of Barley, Wheat, and Rye FPG and OGG1 Genes during Grain Germination.

This research is about the profiling of barley (Hordeum vulgare L.), wheat (Triticum aestivum L.), and rye (Secale cereale L.) FPG and OGG1 genes during grain germination. During seed germination, reactive oxygen species accumulate, which leads to DNA damage. In the base excision repair (BER) system, the enzymes formamidopyrimidine DNA glycosylase (FPG) and 8-oxoguanine DNA glycosylase (OGG1), among others, are responsible for repairing such damage. We decided to check how the expression of genes encoding these two enzymes changes in germinating grains. Spring varieties of barley, wheat, and rye from the previous growing season were used in the study. Expression level changes were checked using Real-Time PCR. After analyzing the obtained results, the maximum expression levels of FPG and OGG1 genes during germination were determined for barley, wheat, and rye. The results of the study show differences in expression levels specific to each species. The highest expression was observed at different time points for each of them. There were no differences in the highest expression for FPG and OGG1 within one species. In conclusion, the research provides information on how the level of FPG and OGG1 gene expression changes during the germination process in cereals. This is the first study looking at the expression levels of these two genes in cereals.

Aluminium(III) Oxide-The Silent Killer of Bacteria.

In this article, we describe the antimicrobial properties of pristine anodised aluminium oxide matrices-the material many consider biologically inert. During a typical anodisation process, chromium and chlorine compounds are used for electropolishing and the removal of the first-step aluminium oxide. Matrices without the use of those harmful compounds were also fabricated and tested for comparison. The antibacterial tests were conducted on four strains of Escherichia coli: K12, R2, R3 and R4. The properties of the matrices were also compared to the three types of antibiotics: ciprofloxacin, bleomycin and cloxacillin using the Minimal Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) tests. Moreover, DNA was isolated from the analysed bacteria which was additionally digested with formamidopyrimidine-DNA glycosylase (Fpg) protein from the group of repair glycosases. These enzymes are markers of modified oxidised bases in nucleic acids produced during oxidative stress in cells. Preliminary cellular studies, MIC and MBC tests and digestion with Fpg protein after modification of bacterial DNA suggest that these compounds may have greater potential as antibacterial agents than the aforementioned antibiotics. The described composites are highly specific for the analysed model Escherichia coli strains and may be used in the future as new substitutes for commonly used antibiotics in clinical and nosocomial infections in the progressing pandemic era. The results show much stronger antibacterial properties of the functionalised membranes on the action of bacterial membranes in comparison to the antibiotics in the Fpg digestion experiment. This is most likely due to the strong induction of oxidative stress in the cell through the breakdown of the analysed bacterial DNA.

Oxidative damage and DNA repair in desiccated recalcitrant embryonic axes of Acer pseudoplatanus L.

BACKGROUND: Most plants encounter water stress at one or more different stages of their life cycle. The maintenance of genetic stability is the integral component of desiccation tolerance that defines the storage ability and long-term survival of seeds. Embryonic axes of desiccation-sensitive recalcitrant seeds of Acer pseudoplatnus L. were used to investigate the genotoxic effect of desiccation. Alkaline single-cell gel electrophoresis (comet assay) methodology was optimized and used to provide unique insights into the onset and repair of DNA strand breaks and 8-oxo-7,8-dihydroguanine (8-oxoG) formation during progressive steps of desiccation and rehydration. RESULTS: The loss of DNA integrity and impairment of damage repair were significant predictors of the viability of embryonic axes. In contrast to the comet assay, automated electrophoresis failed to detect changes in DNA integrity resulting from desiccation. Notably, no significant correlation was observed between hydroxyl radical (Ù OH) production and 8-oxoG formation, although the former is regarded to play a major role in guanine oxidation. CONCLUSIONS: The high-throughput comet assay represents a sensitive tool for monitoring discrete changes in DNA integrity and assessing the viability status in plant germplasm processed for long-term storage.

Functionalised Anodised Aluminium Oxide as a Biocidal Agent.

In this article, we describe the antimicrobial properties of a new composite based on anodic aluminium oxide (AAO) membranes containing propyl-copper-phosphonate units arranged at a predetermined density inside the AAO channels. The samples were prepared with four concentrations of copper ions and tested as antimicrobial drug on four different strains of Escherichia coli (K12, R2, R3 and R4). For comparison, the same strains were tested with three types of antibiotics using the minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) tests. Moreover, DNA was isolated from the analysed bacteria which was additionally digested with formamidopyrimidine-DNA glycosylase (Fpg) protein from the group of repair glycosases. These enzymes are markers of modified oxidised bases in nucleic acids produced during oxidative stress in cells. Preliminary cellular studies, MIC and MBC tests and digestion with Fpg protein after modification of bacterial DNA suggest that these compounds may have greater potential as antibacterial agents than antibiotics such as ciprofloxacin, bleomycin and cloxacillin. The described composites are highly specific for the analysed model Escherichia coli strains and may be used in the future as new substitutes for commonly used antibiotics in clinical and nosocomial infections in the progressing pandemic era. The results show much stronger antibacterial properties of the functionalised membranes on the action of bacterial membranes in comparison to the antibiotics in the Fpg digestion experiment. This is most likely due to the strong induction of oxidative stress in the cell through the breakdown of the analysed bacterial DNA. We have also observed that the intermolecular distances between the functional units play an important role for the antimicrobial properties of the used material. Hence, we utilised the idea of the 2D solvent to tailor them.

Potassium bromate as positive assay control for the Fpg-modified comet assay.

The comet assay is a popular assay in biomonitoring studies. DNA strand breaks (or unspecific DNA lesions) are measured using the standard comet assay. Oxidative stress-generated DNA lesions can be measured by employing DNA repair enzymes to recognise oxidatively damaged DNA. Unfortunately, there has been a tendency to fail to report results from assay controls (or maybe even not to employ assay controls). We believe this might have been due to uncertainty as to what really constitutes a positive control. It should go without saying that a biomonitoring study cannot have a positive control group as it is unethical to expose healthy humans to DNA damaging (and thus potentially carcinogenic) agents. However, it is possible to include assay controls in the analysis (here meant as a cryopreserved sample of cells i.e. included in each experiment as a reference sample). In the present report we tested potassium bromate (KBrO3) as a positive comet assay control for the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay. Ten laboratories used the same procedure for treatment of monocytic THP-1 cells with KBrO3 (0.5, 1.5 and 4.5 mM for 1 h at 37°C) and subsequent cryopreservation. Results from one laboratory were excluded in the statistical analysis because of technical issues in the Fpg-modified comet assay. All other laboratories found a concentration-response relationship in cryopreserved samples (regression coefficients from 0.80 to 0.98), although with different slopes ranging from 1.25 to 11.9 Fpg-sensitive sites (%DNA in tail) per 1 mM KBrO3. Our results demonstrate that KBrO3 is a suitable positive comet assay control.

The genotoxic effects in the leukocytes of workers handling nanocomposite materials.

The extensive development of nanotechnologies and nanomaterials poses a number of questions to toxicologists about the potential health risks of exposure to nanoparticles (NP). In this study, we analysed DNA damage in the leukocytes of 20 workers who were long-term exposed (18 ± 10 years) to NP in their working environment. Blood samples were collected in September 2016, before and after a shift, to assess (i) the chronic effects of NP on DNA (pre-shift samples) and (ii) the acute effects of exposure during the shift (the difference between pre- and post-shift samples). The samples from matched controls were taken in parallel with workers before the shift. Leukocytes were isolated from heparinised blood on a Ficoll gradient. The enzyme-modified comet assay (DNA formamido-pyrimidine-glycosylase and endonuclease III) demonstrated a considerable increase of both single- and double-strand breaks in DNA (DNA-SB) and oxidised bases when compared with the controls (2.4× and 2×, respectively). Acute exposure induced a further increase of DNA-SB. The welding and smelting of nanocomposites represented a higher genotoxic risk than milling and grinding of nanocomposite surfaces. Obesity appeared to be a factor contributing to an increased risk of oxidative damage to DNA. The data also indicated a higher susceptibility of males vs. females to NP exposure. The study was repeated in September 2017. The results exhibited similar trend, but the levels of DNA damage in the exposed subjects were lower compared to previous year. This was probably associated with lower exposure to NP in consequence of changes in nanomaterial composition and working operations. The further study involving also monitoring of personal exposures to NP is necessary to identify (i) the main aerosol components responsible for genotoxic effects in workers handling nanocomposites and (ii) the primary cause of gender differences in response to NP action.

Requirements for DNA bubble structure for efficient cleavage by helix-two-turn-helix DNA glycosylases.

Oxidative DNA lesions, constantly generated by both endogenous and environmentally induced reactive oxygen species, are removed via the base excision repair pathway. In bacteria, Fpg and Nei DNA glycosylases, belonging to the helix-two-turn-helix (H2TH) structural superfamily, remove oxidised purines and pyrimidines, respectively. Interestingly, the human H2TH family glycosylases, NEIL1, NEIL2 and NEIL3, have been reported to prefer oxidative lesions in DNA bubbles or single-stranded DNA. It had been hypothesised that NEIL2 might be involved in the repair of lesions in transcription bubbles; however, bubble-like structures may appear in other cellular contexts such as displacement loops (D-loops) associated with transcription, recombination or telomere maintenance. The activities of bacterial Fpg and Nei on bubble substrates were not addressed. Also, it is not known whether H2TH enzymes process bubbles containing the third DNA or RNA strand, and how the bubble length and position of the lesion within a bubble affect the excision. We have investigated the removal of 8-oxoguanine (8-oxoG) and 5,6-dihydrouracil (DHU) by Escherichia coli Fpg and Nei and human NEIL1 and NEIL2 from single-strand oligonucleotides, perfect duplexes, bubbles with different numbers of unpaired bases (6-30), bubbles containing the lesion in different positions and D-loops with the third strand made of DNA or RNA. Fpg, NEIL1 and NEIL2 efficiently excised lesions located within bubbles, with NEIL1 and NEIL2 being specific for DHU, and Fpg removing both 8-oxoG and DHU. Nei, in contrast, was significantly active only on DHU located in double-stranded DNA. Fpg and NEIL1 also tolerated the presence of the third strand of either DNA or RNA in D-loops if the lesion was in the single-stranded part, and Fpg, Nei and NEIL1 excised lesions from the double-stranded DNA part of D-loops. The presence of an additional unpaired 5'-tail of DNA or RNA did not affect the activity. No significant position preference for lesions in a 12-mer bubble was found. Overall, the activities of Fpg, NEIL1 and NEIL2 on these non-canonical substrates are consistent with the possibility that these enzymes may participate in the repair in structures arising during transcription or homologous recombination.

Critical Sites of DNA Backbone Integrity for Damaged Base Removal by Formamidopyrimidine-DNA Glycosylase.

DNA glycosylases, the enzymes that initiate base excision DNA repair, recognize damaged bases through a series of precisely orchestrated movements. Most glycosylases sharply kink the DNA axis at the lesion site and extrude the target base from the DNA double helix into the enzyme's active site. Little attention has been paid so far to the role of the physical continuity of the DNA backbone in allowing the required conformational distortion. Here, we analyze base excision by formamidopyrimidine-DNA glycosylase (Fpg) from substrates keeping all phosphates but containing a nick within three nucleotides of the lesion in either DNA strand. Four phosphoester linkages at the damaged nucleotide and two nucleotides 3' to it were essential for Fpg activity, while the breakage of the others, even at the same critical phosphates, had no effect or even stimulated the reaction. Reduction of the likelihood of hydrogen bonding at the nicks by using dideoxynucleotides as their 3'-terminal groups was more detrimental for the activity. All phosphoester bonds in the complementary strand were dispensable for base excision, but nicks close to the orphaned nucleotide caused early termination of damaged strand cleavage. Elastic network analysis of Fpg-DNA structures showed that the vibrational motions of the critical phosphates are strongly correlated, in part due to the presence of the protein. Overall, our results suggest that mechanical forces propagating along the DNA backbone play a critical role in the correct conformational distortion of DNA by Fpg and possibly by other target base-everting DNA glycosylases.

A cascade amplification platform assisted with DNAzyme for activity analysis, kinetic study and effector screening of Fpg in vitro.

As a highly conserved damage repair protein, Fpg can specifically recognize and digest 8-oxoG from a damaged DNA backbone. Meanwhile, DNAzyme, a single-stranded DNA with enzymatic activity, can cleave RNA in the presence of cofactors. In this study, we established a highly sensitive method for Fpg assay using a DNAzyme-mediated signal cascade amplification strategy. Based on the Fpg-dependent fluorescence response of the "turn-on" manner, we could reliably determine Fpg activity down to 0.14 U mL-1 with a linear response from 0.10 to 40 U mL-1 under optimal conditions. In addition, this strategy was successfully applied to analyze the kinetic parameter of Fpg with Km of 0.061 µM. Furthermore, the developed sensing system was used to screen the regulators of Fpg from natural compounds and antibiotics. These results indicated that all of the 14 natural compounds and 6 kinds of antibiotics deferentially showed an active effect on Fpg in vitro. In summary, these results show that the method not only provides an alternative for monitoring Fpg activity but also shows great potential for drug screening in the future.

Absolute cross sections for chemoradiation therapy: Damages to cisplatin-DNA complexes induced by 10 eV electrons.

In chemoradiation therapy, the synergy between the radiation and the chemotherapeutic agent (CA) can result in a super-additive treatment. A priori, this increased effectiveness could be estimated from model calculations, if absolute cross sections (ACSs) involved in cellular damage are substantially higher, when the CA binds to DNA. We measure ACSs for damages induced by 10 eV electrons, when DNA binds to the CA cisplatin as in chemotherapy. At this energy, DNA is damaged essentially by the decay of core-excited transient anions into bond-breaking channels. Films of cisplatin-DNA complexes of ratio 5:1 with thicknesses 10, 15, and 20 nm were irradiated in vacuum during 5-30 s. Conformation changes were quantified by electrophoresis and yields extrapolated from exposure-response curves. Base damages (BDs) were revealed and quantified by enzymatic treatment. The ACSs were generated from these yields by two mathematical models. For 3197 base-pair plasmid DNA, ACS for single strand breaks, double strand breaks (DSBs), crosslinks, non-DSB cluster damages, and total BDs is 71 ± 2, 9.3 ± 0.4, 10.1 ± 0.3, 8.2 ± 0.3, and 115 ± 2 ×10-15 cm2, respectively. These ACSs are higher than those of nonmodified DNA by factors of 1.6 ± 0.1, 2.2 ± 0.1, 1.3 ± 0.1, 1.3 ± 0.3, and 2.1 ± 0.4, respectively. Since LEEs are produced in large quantities by radiolysis and strongly interact with biomolecules, we expect such enhancements to produce substantial additional damages in the DNA of the nucleus of cancer cells during concomitant chemoradiation therapy. The increase damage appears sufficiently large to justify more elaborate simulations, which could provide a quantitative evaluation of molecular sensitization by Pt-CAs.

Structural Insight into the Discrimination between 8-Oxoguanine Glycosidic Conformers by DNA Repair Enzymes: A Molecular Dynamics Study of Human Oxoguanine Glycosylase 1 and Formamidopyrimidine-DNA Glycosylase.

hOgg1 and FPG are the primary DNA repair enzymes responsible for removing the major guanine (G) oxidative product, namely, 7,8-dihydro-8-oxoguanine (OG), in humans and bacteria, respectively. While natural G adopts the anti conformation and forms a Watson-Crick pair with cytosine (C), OG can also adopt the syn conformation and form a Hoogsteen pair with adenine (A). hOgg1 removes OG paired with C but is inactive toward the OG:A pair. In contrast, FPG removes OG from OG:C pairs and also exhibits appreciable (although diminished) activity toward OG:A pairs. As a first step toward understanding this difference in activity, we have employed molecular dynamics simulations to examine how the anti and syn conformers of OG are accommodated in the hOgg1 and FPG active sites. When anti-OG is bound, hOgg1 active site residues are properly aligned to initiate catalytic base departure, while geometrical parameters required for the catalytic reaction are not conserved for syn-OG. On the other hand, the FPG catalytic residues are suitably aligned for both OG conformers, with anti-OG being more favorably bound. Thus, our data suggests that the differential ability of hOgg1 and FPG to accommodate the anti- and syn-OG glycosidic conformations is an important factor that contributes to the relative experimental excision rates. Nevertheless, the positions of the nucleophiles with respect to the lesion in the active sites suggest that the reactant complex is poised to initiate catalysis through a similar mechanism for both repair enzymes and supports a recently proposed mechanism in which sugar-ring opening precedes nucleoside deglycosylation.

New Fpg probe chemistry for direct detection of recombinase polymerase amplification on lateral flow strips.

Rapid, cost-effective and sensitive detection of nucleic acids has the ability to improve upon current practices employed for pathogen detection in diagnosis of infectious disease and food testing. Furthermore, if assay complexity can be reduced, nucleic acid amplification tests could be deployed in resource-limited and home use scenarios. In this study, we developed a novel Fpg (Formamidopyrimidine DNA glycosylase) probe chemistry, which allows lateral flow detection of amplification in undiluted recombinase polymerase amplification (RPA) reactions. The prototype nucleic acid lateral flow chemistry was applied to a human genomic target (rs1207445), Campylobacter jejuni 16S rDNA and two genetic markers of the important food pathogen E. coli O157:H7. All four assays have an analytical sensitivity between 10 and 100 copies DNA per amplification. Furthermore, the assay is performed with fewer hands-on steps than using the current RPA Nfo lateral flow method as dilution of amplicon is not required for lateral flow analysis. Due to the simplicity of the workflow, we believe that the lateral flow chemistry for direct detection could be readily adapted to a cost-effective single-use consumable, ideal for use in non-laboratory settings.

Searching for assay controls for the Fpg- and hOGG1-modified comet assay.

The formamidopyrimidine DNA glycosylase (Fpg) and human 8-oxoguanine DNA glycosylase (hOGG1)-modified comet assays have been widely used in human biomonitoring studies. The purpose of this article is to assess differences in reported levels of Fpg- and hOGG1-sensitive sites in leukocytes and suggest suitable assay controls for the measurement of oxidatively damaged DNA. An assessment of the literature showed a large variation in the reported levels of Fpg-sensitive sites (range 0.05-1.31 lesions/106 bp). The levels of Fpg-sensitive sites are lower in studies where Fpg has been obtained from commercial suppliers or unknown sources as compared to Fpg from one particular non-commercial source (χ2 = 7.14, P = 0.028). The levels of hOGG1-sensitive sites are lower (range: 0.04-0.18 lesions/106 bp in leukocytes) compared to the Fpg-sensitive sites. Surprisingly, few publications have reported the use of oxidising agents as assay controls, with the exception of hydrogen peroxide. This may be due to a lack of consensus about suitable controls for the Fpg- and hOGG1-modified comet assay. A major challenge is to find an oxidising agent that only oxidises nucleobases and does not generate DNA strand breaks because this reduces the dynamic range of Fpg- and hOGG1-sensitive sites in the comet assay. Based on a literature search we selected the photosensitiser Ro19-8022 plus light, KBrO3, 4-nitroquinoline-1-oxide, Na2Cr2O7 and ferric nitrilotriacetate as possible assay controls. A subsequent assessment of these compounds for generating cryopreserved assay controls in mononuclear blood cells showed that Ro19-8022 plus light, KBrO3 and 4-nitroquinoline-1-oxide provided suitable assay controls. We recommend these compounds as comet assay controls for oxidatively damaged DNA.

The comet assay in human biomonitoring: cryopreservation of whole blood and comparison with isolated mononuclear cells.

The comet assay is often applied in human biomonitoring. Most of the time the assay is performed with isolated peripheral blood mononuclear cells (PBMC). However, using whole blood instead of isolated cells reduces processing time, and only 20 µl is sufficient for analysis. In this study, a cryopreservation protocol for human whole blood for application in the comet assay was optimised by removing excess plasma before adding freezing medium. Cryopreservation of whole blood samples (n = 30) did not increase the detected level of strand breaks and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites. Although there was no significant correlation with breaks measured in fresh whole blood, strand breaks detected in frozen whole blood were significantly correlated with breaks measured in frozen PBMC (Pearson correlation r = 0.54, P < 0.01). This correlation was however not observed for FPG-sensitive sites. Since we do not yet know the full extent to which cryopreservation might influence the blood cell population, care should be taken to ensure a similar cell type and storage conditions for all samples in one study.

Using the comet assay and lysis conditions to characterize DNA lesions from the acrylamide metabolite glycidamide.

The alkaline comet assay and a cell-free system were used to characterise DNA lesions induced by treatment with glycidamide (GA), a metabolite of the food contaminant acrylamide. DNA lesions induced by GA were sensitively detected when the formamidopyrimidine-DNA-glycosylase (Fpg) enzyme was included in the comet assay. We used LC-MS to characterise modified bases from GA-treated naked DNA with and without subsequent Fpg treatment. N7-GA-Guanine and N3-GA-Adenine aglycons were detected in the supernatant showing some depurination of adducted bases; treatment of naked DNA with Fpg revealed no further increase in the adduct yield nor occurrence of other adducted nucleobases. We treated human lymphocytes with GA and found large differences in DNA lesion levels detected with Fpg, depending on the duration and the pH of the lysis step. These lysis-dependent variations in GA-induced Fpg sensitive sites paralleled those observed after treatment of cells with methyl methane sulfonate (MMS). On the other hand, oxidative lesions (8-oxoGuanine) induced by a photoactive compound (Ro 12-9786) plus light, and also DNA strand breaks induced by X-rays, were detected largely independently of the lysis conditions. The results suggest that the GA-induced lesions are predominantly N7-GA-dG adducts slowly undergoing imidazole ring opening at pH 10 as in the standard lysis procedure; such structures are substrate for Fpg leading to strand breaks. The data suggest that the characteristic alkaline lysis dependence of some DNA lesions may be used to study specific types of DNA modifications. The comet assay is increasingly used in regulatory testing of chemicals; in this context, lysis-dependent variations represent a novel approach to obtain insight in the molecular nature of a genotoxic insult.