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A number of new conjugates of dehydroabiethylamine and adamantane was obtained using a three-step synthetic scheme in order to study the effect of linker type and length on biological activity. The inhibitory activity of the synthesized compounds toward the DNA repair enzyme Tyrosyl-DNA phosphodiesterase 1 was studied. The compounds are potent inhibitors, exhibiting activity in micromolar concentrations. [ABSTRACT FROM AUTHOR]

The human genome is continually exposed to various stressors, which can result in DNA damage, mutations, and diseases. Among the different types of DNA damage, single-strand lesions are commonly induced by external stressors and metabolic processes. Accurate detection and quantification of DNA damage are crucial for understanding repair mechanisms, assessing environmental impacts, and evaluating response to therapy. However, traditional techniques have limitations in sensitivity and the ability to detect multiple types of damage. In recent years, single-molecule fluorescence approaches have emerged as powerful tools for precisely localizing and quantifying DNA damage. Repair Assisted Damage Detection (RADD) is a single-molecule technique that employs specific repair enzymes to excise damaged bases and incorporates fluorescently labeled nucleotides to visualize the damage. This technique provides valuable insights into repair efficiency and sequence-specific damage. In this review, we discuss the principles and applications of RADD assays, highlighting their potential for enhancing our understanding of DNA damage and repair processes. • Single-strand DNA damage is the most common yet the least explored form of DNA damage. • Single-molecule fluorescence assays provide the ultimate sensitivity for damage detection by counting individual lesions along extended DNA molecules. • Repair enzyme cocktails used for DNA damage detection may be tailored to label specific damage lesions or combined for broad spectrum detection. • Tracking the number of damage sites over time provides valuable insights into cell repair dynamics. • DNA damage may be mapped to specific genomic loci by combining RADD with optical genome mapping approaches. [ABSTRACT FROM AUTHOR]

As part of the EU-project COMICS, high throughput versions of the comet assay was developed. In order to test the performance of the new formats; the novel 12-gel slide and the 96-minigel film were compared with standard 3-gel slides for their ability to detect effects of genotoxic chemicals on cellular DNA with limited cytotoxicity. Among the chemicals were negative controls, a non-carcinogen that causes DNA damage through cytotoxicity, and carcinogens that are known to be hard to detect by simple DNA strand break analysis. Chemicals requiring metabolic activation were preincubated with rat liver S9 fraction. TK-6 human lymphoblastoid cells were treated with a range of concentrations of each chemical, above and below the expected cytotoxic concentration. Trials were carried out in 3 centres applying all three formats. Results obtained with the three systems (standard, medium- and high-throughput) were essentially the same. The 96-minigel format was analysed with the fully automated scoring system IMSTAR and comparable results were achieved with the semi-automated scoring system from Perceptives. The known genotoxic chemicals MNU, B(a)P, 4-NQO and cyclophosphamide showed little consistent sign of genotoxicity at concentrations causing limited cytotoxicity. D-mannitol and Triton X-100 were, as expected, non-genotoxic (though Triton X-100, at high concentrations, caused DNA breaks as an apparent secondary effect of cytotoxicity). Etoposide and bleomycin gave significant increase in DNA strand break at borderline cytotoxic concentrations. The limitation of the assay to detect damaged bases by known genotoxins may be overcome by incorporating a DNA repair enzyme, such as formamidopyrimidine-DNA-glycosylase (FPG), to convert damaged bases into breaks as shown by Azqueta A et al., Mutagenesis vol. 28 no. 3 pp. 271–277, 2013 .

Having their position gained among the smallest bulky DNA lesions recognized by the nucleotide excision repair (NER) enzyme, purine 5′,8-cyclo-2′-deoxynucleosides (5′,8-cPu) are increasingly attracting the interest in the field of genome integrity in health and diseases. Exclusively generated by one of the most harmful of the reactive oxygen species, the hydroxyl radical, 5′,8-cPu can be utilized also for highly valuable information regarding the oxidative status nearby the area where the genetic information is stored. Herein, we have collected the most recently reported biological studies, focusing on the repair mechanism of these lesions and their biological significance particularly in transcription. The LC-MS/MS quantification protocols that appeared in the literature are discussed in details, along with the reported values for the four 5′,8-cPu produced by in vitro γ-radiolysis experiments with calf thymus DNA. Mechanistic insights in the formation of the purine 5′,8-cyclo-2′-deoxynucleosides and their chemical stability are also given in the light of their potential to be utilized as DNA biomarkers of oxidative stress. [ABSTRACT FROM AUTHOR]

The article discusses a laboratory experiment which examined the effects of increased temperatures and ions on the stability of DNA at different acidity (pH) levels, based on either Bacillus subtilis and Escherichia coli. Several changes were detected under these conditions including susceptibility to hydrolytic attack, oxidative damage and alkylation at guanine, cytosine, thymine and adenine sites. The role of antibody-producing cells in changing the DNA structure is mentioned.

The global burden of cardiovascular disease is increasing despite therapeutic advances in medication and interventional technologies. Accumulated deoxyribonucleic acid (DNA) damage and subsequent repair pathways are now increasingly recognised as a causal factor in the initiation and progression of atherosclerosis. These molecular alterations have been shown to occur within affected vasculature, plaque microenvironment as well as in circulating cells. The DNA damage response (DDR) pathway is reliant on post-translational modification of sensing proteins which activate a signalling cascade to repair, if possible, DNA damaged sites in response to various environmental and physiological insults. This review summarises the current evidence for DNA damage in atherosclerosis, the key steps involved in the DDR pathway, DNA repair and their subsequent effects on atherosclerotic plaques, as well as the therapeutic options in managing DNA damage-induced atherosclerosis. [ABSTRACT FROM AUTHOR]

Background: Gastric cancer results from a multifactorial process and is one of the most common causes of death worldwide. These tumors can arise in the distal stomach (non-cardia) and in the cardia region, presenting different characteristics and frequency of occurrence worldwide. Aims: To search for differences between tumors of different locations that could explain the presence of cardia tumors, considering Helicobacter pylori strains and genetic polymorphisms. Materials and Methods: DNA was extracted from gastric adenocarcinoma tissue of 127 patients. Helicobacter pylori genes were detected by PCR, and polymorphisms by PCR-RFLP. Results: Most of the tumors were located in non-cardia. The genotype 28152GA of XRCC1 showed an increase in risk of cardia tumors. In analysis performed considering gender, women carrying TNF-308GA genotype showed a decreased risk of non-cardia tumors, while in men the decreased risk of non-cardia tumors was associated with TNF-308GG genotype. Genotypes combinations showed that the SNPs RAD51 135G>C, XRCC3 18067C>T, and XRCC1 28152G>A had some combinations more frequent in cardia tumors, with an increased risk. Patients infected by cagE-positive strains presented a positive correlation with non-cardia tumors. Conclusion: The results showed some susceptibility differences between tumors of different locations. There was an increased risk relationship between three repair enzyme SNPs and cardia tumors, and the G allele of the cytokine gene TNF negatively influenced the development of non-cardia tumors. Helicobacter pylori strains seemed to be different in the cardia region, where they were less virulent than those located in the distal region of the stomach. [ABSTRACT FROM AUTHOR]

Clustered DNA damage induced by 10, 20 and 30 MeV protons in pBR322 plasmid DNA was investigated. Besides determination of strand breaks, additional lesions were detected using base excision repair enzymes. The plasmid was irradiated in dry form, where indirect radiation effects were almost fully suppressed, and in water solution containing only minimal residual radical scavenger. Simultaneous irradiation of the plasmid DNA in the dry form and in the solution demonstrated the contribution of the indirect effect as prevalent. The damage composition slightly differed when comparing the results for liquid and dry samples. The obtained data were also subjected to analysis concerning different methodological approaches, particularly the influence of irradiation geometry, models used for calculation of strand break yields and interpretation of the strand breaks detected with the enzymes. It was shown that these parameters strongly affect the results. [ABSTRACT FROM AUTHOR]

Specific and nonspecific DNA complex formation with human uracil-DNA glycosylase, 8-oxoguanine-DNA glycosylase, and apurine/apyrimidine endonuclease, as well as with E. coli 8-oxoguanine-DNA glycosylase and RecA protein was analyzed using the method of stepwise increase in DNA-ligand complexity. It is shown that high affinity of these enzymes to any DNA (10-10 M) is provided by a large number of weak additive contacts mainly with DNA internucleoside phosphate groups and in a less degree with bases of nucleotide links 'covered' by protein globules. Enzyme interactions with specific DNA links are comparable in efficiency with weak unspecific contacts and provide only for one-two orders of affinity (10-10 M), but these contacts are extremely important at stages of DNA and enzyme structural adaptation and catalysis proper. Only in the case of specific DNA individual for each enzyme alterations in DNA structure provide for efficient adjustment of reacting enzyme atoms and DNA orbitals with accuracy up to 10-15° and, as a result, for high reaction rate. Upon transition from nonspecific to specific DNA, reaction rate ( k) increases by 4-8 orders of magnitude. Thus, stages of DNA and enzyme structural adaptation as well as catalysis proper are the basis of specificity of repair enzymes. [ABSTRACT FROM AUTHOR]

A study was made of the interaction of 8-oxoguanine-DNA glycosylases of Escherichia coli (Fpg) and human (OGG1), as well as apurinic/apyrimidinic endonucleases of yeast (Apn1) and E. coli (Nfo), with oligodeoxyribonucleotides containing 8-oxoguaine (oxoG) and tetrahydrofuran (F, a stable analog of an apurinic site) separated by various numbers of nucleotides. Inhibitor analysis showed that the affinity of Fgp for single-stranded DNA ligands is virtually independent of the relative positions of oxoG and F. K M and k cat were determined for all the four enzymes and all double-stranded substrates studied. The effect of the second lesion strongly depended both on the relative position of the lesion and the enzyme of interest. The highest drop in the affinity of Fpg and OGG1 for the substrate (1.6-to 148-fold) and in the reaction rate (4.8-to 58-fold) was recorded for the oligonucleotides in which F was immediately 3′ or 5′ of oxoG. Introduction of the second lesion barely affected K M for nucleases Apn1 and Nfo. The reaction rate was five-to tenfold lower for the substrates containing two adjacent lesions. For all enzymes studied, an increase in the distance between two lesions in double-stranded DNA decreased their contribution to K M and k cat. [ABSTRACT FROM AUTHOR]

Proteins are subject to modification by reactive oxygen species (ROS), and oxidation of specific amino acid residues can impair their biological function, leading to an alteration in cellular homeostasis. Methionine is among the amino acids the most susceptible to oxidation by almost all forms of ROS, resulting in both S and R diasteroisomeric forms of methionine sulfoxide. These modifications can be repaired specifically by the peptide methionine sulfoxide reductase A and B enzymes (MsrA and MsrB), respectively. MsrA has been detected in several organisms going from prokaryotes to eukaryotes. MsrA is tightly implicated in protection against oxidative stress and in protein maintenance, which is critical in the aging process. Several studies have shown that overexpression of MsrA led to an increased resistance against oxidative stress, while MsrA null mutants are more sensitive toward oxidative stress. Since oxidative damage is a key factor in aging, overexpression of MsrA in some organisms led to an increased life span whereas deletion of the gene led to the opposite. MsrA could also be involved, by regulating the function and/or expression of target proteins, in ROS-mediated signal transduction. In fact, changes in gene expression, including certain oxidative stress–response genes, have been observed when MsrA is overexpressed. This review elaborates on the current knowledge in the implication of the Msr system in protection against oxidative stress and aging. [ABSTRACT FROM AUTHOR]

Bleomycin is a small glycopeptide antibiotic used in combination therapy for the treatment of a few types of human cancer. The antitumor effect of bleomycin is most likely caused by its ability to bind to DNA and induce the formation of toxic DNA lesions via a free radical reactive (Fe.bleomycin) complex. However, the chemotherapeutic potential of bleomycin is limited, as it causes pulmonary fibrosis and tumor resistance at high doses. The chemical structure and modes of action of bleomycin have been extensively studied and these provide a foundation towards improving the therapeutic value of the drug. This review provides a first account of the current state of knowledge of the cellular processes that can allow the yeast Saccharomyces cerevisiae to evade the lethal effects of bleomycin. This model organism is likely to provide rapid clues in our understanding of bleomycin resistance in tumor cells. [ABSTRACT FROM AUTHOR]

Molecular dynamics (MD) studies of several radiation originated lesions on the DNA molecules are presented. The pyrimidine lesions (cytosinyl radical, thymine dimer, thymine glycol) and purine lesion (8-oxoguanine) were subjected to the MD simulations for several hundred picoseconds using MD simulation code AMBER 5.0 (4.0). The simulations were performed for fully dissolved solute molecules in water. Significant structural changes in the DNA double helical structure were observed in all cases which may be categorized as: a) the breaking of hydrogen bonds network between complementary bases and resulted opening of the double helix (cytosinyl, radical, 8-oxoguanine); b) the sharp bending of the DNA helix centered at the lesion site (thymine dimer, thymine glycol); and c) the flippingout of adenine on the strand complementary to the lesion (8-oxoguanine). These changes related to the overall collapsing of the double helical structure around the lesion, are expected to facilitate the docking of the repair enzyme into the DNA in the formation of DNA-enzyme complex. The stable DNA-enzyme complex is a necessary condition for the onset of the enzymatic repair process. In addition to structural changes, specific values of electrostatic interaction energy were determined at several lesion sites (thymine dimer, thymine glycol and 8-oxoguanine). This lesion-specific electrostatic energy is a factor that enables repair enzyme to discriminate lesion from the native site during the scanning of the DNA surface. [ABSTRACT FROM AUTHOR]

It has been reported that on reaching a certain level of cell damage the production of repair enzymes is triggered which decreases the chromosome aberrations. If this happens, prolonged exposure to high levels of natural radiation in areas with elevated levels of background radiation could decrease the frequency of chromosome aberrations. Recent epidemiological studies indicated that there is an increased risk of cancer in healthy individuals with high levels of chromosomal aberrations. Studies performed in Nordic countries as well as Italy, showed that increased levels of chromosome aberrations in lymphocytes can be used to predict cancer risk in humans. One may conclude that a dose of ionising radiation sufficient to produce a certain level of cell damage increases production of antioxidants and repair enzymes that decrease either the frequency of chromosome aberrations or the cancer risk. People in some areas of Ramsar, a city in northern Iran, receive an annual radiation dose from background radiation that is more than five times higher than the 20 mSv. Yr-1 that is permitted for radiation workers. Inhabitants of Ramsar have lived for many generations in these high background areas. If an annual radiation dose of a few hundred mSv is detrimental to health, causing genetic abnormalities or an increased risk of cancer, it should be evident in these people. The absorbed dose rate in some high background radiation areas of Ramsar is approximately 55-200 times higher than that of the average global dose rate. It has been reported that 3-8% of all cancers are caused by current levels of ionising radiation. If this estimation were true, all the inhabitants of such an area with extraordinary elevated levels of natural radiation would have died of cancer. Our cytogenetic studies show no significant differences between people in the high background area compared to people in normal background areas. As there was no increased level of chromosome aberrations, it may be predicted that the cancer incidence is not higher than in the neighbouring areas with a normal background radiation level. Although there is not yet solid epidemiological information, most local physicians in Ramsar report anecdotally that there is no increase in the incidence rates of cancer or leukemia in their area. There are no data to indicate a significant increase of cancer incidence in other high background radiation areas (HBRAs). Furthermore, several studies show a significant decrease of cancer death rates in areas with high backgrounds. It can be concluded that prolonged exposure to high levels of natural radiation possibly triggers processes such as the production of antioxidants and repair enzymes, which decreases the frequency of chromosome aberrations and the cancer incidence rate. [ABSTRACT FROM AUTHOR]

Presents an abstract of a study on cancer incidence in areas with elevated levels of natural radiation. Effect of the prolonged exposure to high levels of natural radiation on the frequency of chromosome aberrations; Medical condition that can be used to predict cancer risk in humans; Human biological process, which decreases the frequency of chromosome aberrations and the cancer incidence rate.

The purpose of the present review is to summarize the data related with the structural features of interaction between the human repair enzyme 8-oxoguanine DNA glycosylase (hOGG1) and DNA. The review covers the questions concerning the role of individual amino acids of hOGG1 in the specific recognition of the oxidized DNA bases, formation of the enzyme-substrate complex, and excision of the lesion bases from DNA. Attention is also focused upon conformational changes in the enzyme active site and disruption of enzyme activity as a result of amino acid mutations. The mechanism of damaged bases release from DNA induced by hOGG1 is discussed in the context of structural dynamics.