Resveratrol and ascorbic acid prevent DNA damage induced by cryopreservation in human semen

Cryopreservation of human semen can cause DNA damages, which compromise the fertilization and normal embryo development. The present study showed that the antioxidant resveratrol prevents these damages both in fertile and infertile men. The addition of ascorbic acid before cryopreservation can reduce DNA damages only in infertile men. Although further studies are needed, the present work showed that resveratrol could be considered in human cryopreservation procedures to avoid/minimize DNA damages and preserve sperm integrity.     

A new calculation on spectrum of direct DNA damage induced by low-energy electrons

In this work, direct DNA damage induced by low-energy electrons (<5 keV) is simulated using Monte Carlo methods, and the resulting yield of various strand breaks and base damages in cellular environment is presented. The simulation is based on a new inelastic cross section for the production of electron track structure in liquid water, and on ionization cross sections of DNA bases to generate base radical. Especially, a systematic approach of simulating detailed base damage is suggested. This approach includes improvement of a volume model of DNA, generation of the DNA base sequence, conversion of ionization events in liquid water at hit site to the ionization interaction of electrons with DNA bases and development of an algorithm to convert a base radical to a damage. The results obtained in terms of strand breaks are compared with those of experiments and other theoretical calculations, and good agreement was obtained. The yield of detailed base damages and clustered DNA damages caused by the combination of various strand breaks and base damages is presented, and the corresponding distribution characteristics are analyzed. The influence of the relative content of base pairs A-T and G-C in a DNA segment on the yield of both strand breaks and base damages is also explored. The present work provides fundamental information on DNA damage and represents the first effort toward the goal of obtaining the spectrum of clustered DNA damage including detailed base damages, for the mechanistic interpretation and prediction of radiation effects.     

Low efficiency of repair of critical DNA damage induced by low doses of radiation

This study provides an analysis of the development of cellular response to the critical DNA damage and the mechanisms for limiting the efficiency of repairing such damages induced by low doses of ionizing radiation exposure. Based on the data of many studies, one can conclude that the majority of damages occurring in the DNA of the cells after exposure to ionizing radiation significantly differ in their chemical nature from the endogenous ones. The most important characteristic of radiation-induced DNA damages is their complexity and clustering. Double strand breaks, interstrand crosslinks or destruction of the replication fork and formation of long single-stranded gaps in DNA are considered to be critical damages for the fate of cells. The occurrence of such lesions in DNA may be a key event in the etiology and the therapy of cancer. The appearance in the cells of the critical DNA damage induces a rapid development of a complex and ramified network of molecular and biochemical reactions which are called the cellular response to DNA damage. Induction of the cellular response to DNA damage involves the activation of the systems of cell cycle checkpoints, DNA repair, changes in the expression of many genes, reconstruction of the chromatin or apoptosis. However, the efficiency of repair of the complex DNA damage in cells after exposure to low doses of radiation remains at low levels. The development of the cell response to DNA damages after exposure to low doses of radiation does not reach the desired result due to a small amount of damage, with the progression of the phase cell cycle being ahead of the processes of DNA repair. This is primarily due to the failure of signalization to activate the checkpoint of the cell cycle for its arrest in the case of a small number of critical DNA lesions. In the absence of the arrest of the phase cell cycle progression, especially during the G2/M transition, the reparation mechanisms fail to completely restore DNA, and cells pass into mitosis with a damaged DNA. It is assumed that another reason for the low efficiency of DNA repair in the cells after exposure to low doses of radiation is the existence of a restricted access for the repair system components to the complex damages at the DNA sites of highly compacted chromatin.     

Quantifying clustered DNA damage induction and repair by gel electrophoresis, electronic imaging and number average length analysis

Assessing DNA damage induction, repair and consequences of such damages requires measurement of specific DNA lesions by methods that are independent of biological responses to such lesions. Lesions affecting one DNA strand (altered bases, abasic sites, single strand breaks (SSB)) as well as damages affecting both strands (clustered damages, double strand breaks) can be quantified by direct measurement of DNA using gel electrophoresis, gel imaging and number average length analysis. Damage frequencies as low as a few sites per gigabase pair (10(9)bp) can be quantified by this approach in about 50ng of non-radioactive DNA, and single molecule methods may allow such measurements in DNA from single cells. This review presents the theoretical basis, biochemical requirements and practical aspects of this approach, and shows examples of their applications in identification and quantitation of complex clustered damages.     

Role of the processes of repair of DNA damage in the repair of cytogenetic disorders. Participation of the repair of DNA single-strand breaks in the repair of cytogenetic disorders

A comparison was made between the results of the effect of poly(ADP-ribosylation) inhibitors (e.g. nicotinamide and 3-aminobenzamide) and a chromatin proteinase inhibitor, phenylmethylsulfonylfluoride, on the cytogenetic damages repair, by a micronucleus test and DNA repair in Chinese hamster fibroblasts. The values of the repair half-periods (5-7 min for the cytogenetic damages and 5 min for the rapidly repaired DNA damages) and a similar modifying effect with regard to radiation cytogenetic damages and kinetics of DNA damages repair were found to be close. This confirms the contribution of repair of DNA single-strand breaks in the initiation of structural damages to chromosomes.     

Modeling radiation damage to DNA using radionuclides incorporated into polynucleotides

Because of a great variety and different reparability of radiation-induced DNA lesions it is difficult to evaluate the radiobiological significance of certain individual alterations. It is suggested that the radionuclides incorporated into DNA can be used to imitate different types of radiation damages to DNA. Both qualitative and quantitative aspects of the problem are discussed.     

Heavy ion-induced plasmid DNA damage in aerated or deaerated conditions

Using the plasmid relaxation assay, the induction of single strand breaks (SSB) and base damages was investigated in air-dried plasmid DNA irradiated under air or under vacuum, with two high LET particles. We first observed that an irradiation with 12C5+ ion produced less of both damages when performed in a vacuum rather than in the presence of air. This could be due to the presence of O2 which increases the primary radicalar effects in the latter case. Another explanation is a difference in the degree of hydration of the DNA molecules. Indeed, under vacuum only the water molecules tightly bound to DNA will persist. In contrast, in the presence of air, the outer hydration shell enhances the amount of hydroxyl radicals available for the radiolytic attack. However, no difference in the SSB induction was observed when DNA was irradiated with 36S16+ ion in the presence of air or under vacuum. This is likely due to the LET effect which partly cancels the production of radicals by recombination and increases the formation of superoxide anions in the track. Similarly, the lower induction of damage by 36S16+ irradiation in comparison with the 12C5+ ion is a consequence of the higher ionizing density for 36S16+ than for 12C5+ ions. Meanwhile, for both ions, base damages are not detected when DNA is irradiated under vacuum, whereas they are as frequent as SSB when irradiation is performed in the presence of air. Altogether, these observations support the idea that SSB and base damage are not formed by the same mechanism.     

Cellular role of yeast Apn1 apurinic endonuclease/3''-diesterase: repair of oxidative and alkylation DNA damage and control of spontaneous mutation.

The APN1 gene of Saccharomyces cerevisiae encodes the major apurinic/apyrimidinic endonuclease and 3'-repair DNA diesterase in yeast cell extracts. The Apn1 protein is a homolog of Escherichia coli endonuclease IV, which functions in the repair of some oxidative and alkylation damages in that organism. We show here that yeast strains lacking Apn1 (generated by targeted gene disruption or deletion-replacement) are hypersensitive to both oxidative (hydrogen peroxide and t-butylhydroperoxide) and alkylating (methyl- and ethylmethane sulfonate) agents that damage DNA. These cellular hypersensitivities are correlated with the accumulation of unrepaired damages in the chromosomal DNA of apn1 mutant yeast cells. Hydrogen peroxide-treated APN1+ but not apn1 mutant cells regenerate high-molecular-weight DNA efficiently after the treatment. The DNA strand breaks that accumulate in the Apn1-deficient mutant contain lesions that block the action of DNA polymerase but can be removed in vitro by purified Apn1. An analogous result with DNA from methylmethane sulfonate-treated cells corresponded to the accumulation of unrepaired DNA apurinic sites in the apn1 mutant cells. The rate of spontaneous mutation in apn1 mutant S. cerevisiae was 6- to 12-fold higher than that measured for wild-type yeast cells. This increase indicates that under normal growth conditions, the production of DNA damages that are targets for Apn1 is substantial and that such lesions can be mutagenic when left unrepaired.     

Modulation of DNA damage/DNA repair capacity by XPC polymorphisms

XPC, a key protein in the nucleotide excision repair (NER) pathway, recognizes damaged DNA and initiates NER. Genetic variations in the XPC gene might be associated with altered DNA repair capacities (DRC). In this study, we genotyped three XPC polymorphisms, Ala499Val (C-->T), PAT (-/+) and Lys939Gln (A-->C), and measured the DNA damage/DRC by alkaline comet assay challenged by BPDE and gamma-radiation in 476 healthy subjects. We also evaluated the associations between DNA damage/DRC and genotypes of XPC polymorphisms. Compared with the XPC Lys939Gln homozygous wild type (AA) subjects, subjects with the variant alleles (AC and CC) had significantly higher DNA damages induced by BPDE (Median and 95% confidence interval [CI]: 3.16 (3.01-3.44) vs. 2.88 (2.51-3.05), P=0.01), and gamma-radiation (4.18 (3.94-4.44) vs. 3.71 (3.49-4.04), P=0.01). However, subjects with the variant alleles (CT and TT) of Ala499Val exhibited a 8.6% and 13.1% decrease in DNA damages induced by BPDE (P=0.05) and gamma-radiation (P=0.001), respectively. Significant correlations were found between genotypes and induced DNA damages in XPC Lys939Gln (For BPDE: R=0.12, P=0.01; for gamma-radiation: R=0.094, P=0.046) and Ala499Val (For BPDE: R=-0.11, P=0.03; for gamma-radiation: R=-0.16, P=0.0009). The haplotypes "T-A" (in the order of Ala499Val-PAT-Lys939Gln) was associated with the lowest DNA damages. Our results suggested that the DRC of host cells might be modulated by specific XPC polymorphisms.     

The role of DNA repair processes in the repair of cytogenetic disorders. The participation of slow to repair DNA damage in the repair of cytogenetic disorders

Caffeine was used to study the kinetics of cytogenetic damages repair in Chinese hamster fibroblasts. Its half-time (90 min) was shown to correlate with that of repair of slowly repaired DNA damages. The caffeine-induced increase in the number of irreparable DNA damages, attributed to inhibition of double-strand break repair, is in a quantitative correlation with the effect of the cytogenetic damage modification.     

Gene regulation in response to DNA damage

To deal with different kinds of DNA damages, there are a number of repair pathways that must be carefully orchestrated to guarantee genomic stability. Many proteins that play a role in DNA repair are involved in multiple pathways and need to be tightly regulated to conduct the functions required for efficient repair of different DNA damage types, such as double strand breaks or DNA crosslinks caused by radiation or genotoxins. While most of the factors involved in DNA repair are conserved throughout the different kingdoms, recent results have shown that the regulation of their expression is variable between different organisms. In the following paper, we give an overview of what is currently known about regulating factors and gene expression in response to DNA damage and put this knowledge in context with the different DNA repair pathways in plants. This article is part of a Special Issue entitled: Plant gene regulation in response to abiotic stress.     

Excision of 8-oxoguanine within clustered damage by the yeast OGG1 protein

Clustered damages are formed in DNA by ionising radiation and radiomimetic anticancer agents and are thought to be biologically severe. 7,8-dihydro-8-oxoguanine (8-oxoG), a major DNA damage resulting from oxidative attack, is highly mutagenic leading to a high level of G·C→T·A transversions if not previously excised by OGG1 DNA glycosylase/AP lyase proteins in eukaryotes. However, 8-oxoG within clustered DNA damage may present a challenge to the repair machinery of the cell. The ability of yeast OGG1 to excise 8-oxoG was determined when another type of damage [dihydrothymine, uracil, 8-oxoG, abasic (AP) site or various types of single-strand breaks (SSBs)] is present on the complementary strand 1, 3 or 5 bases 5′ or 3′ opposite to 8-oxoG. Base damages have little or no influence on the excision of 8-oxoG by yeast OGG1 (yOGG1) whereas an AP site has a strong inhibitory effect. Various types of SSBs, obtained using either oligonucleotides with 3′- and 5′-phosphate termini around a gap or through conversion of an AP site with either endonuclease III or human AP endonuclease 1, strongly inhibit excision of 8-oxoG by yOGG1. Therefore, this large inhibitory effect of an AP site or a SSB may minimise the probability of formation of a double-strand break in the processing of 8-oxoG within clustered damages.     

DNMT3b protects centromere integrity by restricting R-loop-mediated DNA damage

This study used DNA methyltransferase 3b (DNMT3b) knockout cells and the functional loss of DNMT3b mutation in immunodeficiency-centromeric instability-facial anomalies syndrome (ICF) cells to understand how DNMT3b dysfunction causes genome instability. We demonstrated that R-loops contribute to DNA damages in DNMT3b knockout and ICF cells. More prominent DNA damage signal in DNMT3b knockout cells was due to the loss of DNMT3b expression and the acquirement of p53 mutation. Genome-wide ChIP-sequencing mapped DNA damage sites at satellite repetitive DNA sequences including (peri-)centromere regions. However, the steady-state levels of (peri-)centromeric R-loops were reduced in DNMT3b knockout and ICF cells. Our analysis indicates that XPG and XPF endonucleases-mediated cleavages remove (peri-)centromeric R-loops to generate DNA beaks, causing chromosome instability. DNMT3b dysfunctions clearly increase R-loops susceptibility to the cleavage process. Finally, we showed that DNA double-strand breaks (DSBs) in centromere are probably repaired by error-prone end-joining pathway in ICF cells. Thus, DNMT3 dysfunctions undermine the integrity of centromere by R-loop-mediated DNA damages and repair.Subject terms: Double-strand DNA breaks, DNA methylation, Primary immunodeficiency disorders     

白念珠菌DNA损伤与修复

内外环境中各种因素如电离辐射、紫外辐射、氧化剂、烷化剂等都可以造成白念珠菌DNA的损伤。如果DNA的损伤得不到有效的修复,便会造成突变。白念珠菌的突变率很高,但并不是所有DNA受损伤的细胞都会表现出突变型性状,这跟其自身的修复系统有很大关系,主要包括切除修复、错配修复及双链断裂修复等途径,使得绝大多数损伤能够及时修复,从而维持DNA的完整性与稳定性。白念珠菌DNA的损伤修复可能影响其适应性、药物敏感性等表型,从而给临床感染患者的治疗增加难度。本文主要从白念珠菌DNA损伤的产生,损伤信号的传导识别及损伤修复三方面综述目前的研究进展。     

Long-term DNA damage and mammalian cell survival

On the basis of our own data and those reported in the literature we have made an attempt to follow the fate of the DNA lesions which remain unrepaired during a long period of time, and their possible role in the fate of irradiated cells. The presence of long-lived ("residual") damages is determined by the changes in survival of exposed cells treated, at different times after irradiation, with a mixture of arabinoside cytosine and hydroxyurea. It is shown that "residual" damages can probably exist in the exposed generation and be retained in that following the irradiated one, i.e. after the first mitosis. The nearest descendants of exposed cells (the 3d-5th generations) exhibit a 50% decrease in the rate of DNA synthesis and fall of their proliferative activity, as well as a decrease in the rate of reproduction of their remote descendants. The comparison of the results obtained with those reported by other authors enable us to assume that "residual" DNA lesions play an important role in the fate of exposed cells, that is, in reproductive death, radiation mutagenesis, and malignant transformations.     

Evidences showing ultraviolet-B radiation-induced damage of DNA in cyanobacteria and its detection by PCR assay

Impact of ultraviolet-B radiation in causing the damages to the DNA of the cyanobacterium, Anabaena strain BT2 has been investigated. Exposure of genomic DNA (in vitro) to UV-B radiation for 1 h did not cause any shift in the absorption peak (lambda(max)) but more than 30% increase in absorbance was noticed in comparison to untreated control DNA (no exposure to UV-B). This increase in absorbance in a way may be comparable to typical hypochromic effect but there was no decrease in absorbance following transfer of UV-B-treated DNA to fluorescent light or in the dark. That the damaging effect of UV-B radiation on native structure of DNA is indeed real was also evident from the PCR-based assay such as RAPD, rDNA amplification, and ARDRA. Template activity of UV-B-treated genomic DNA was drastically inhibited, there was no amplification in RAPD assay after prior exposure of DNA to UV-B for 60 min. Only one band of approximately 400 bp was observed even after 60 min of exposure which suggests that certain segment of DNA strand is resistant to UV-B effects. Similar to the effects on RAPD profile, amplification of rDNA was significantly inhibited following exposure of genomic DNA to UV-B. Our findings clearly demonstrate that UV-B does affect the DNA of cyanobacteria and the killings of these microbes might be due to the irreversible damages caused to DNA by this high energy radiation. It is felt that PCR assay may be conveniently used for screening the damages caused to DNA by UV-B radiation in cyanobacteria and other microorganisms.     

L-carnosine reduces telomere damage and shortening rate in cultured normal fibroblasts

Telomere is the repetitive DNA sequence at the end of chromosomes, which shortens progressively with cell division and limits the replicative potential of normal human somatic cells. L-carnosine, a naturally occurring dipeptide, has been reported to delay the replicative senescence, and extend the lifespan of cultured human diploid fibroblasts. In this work, we studied the effect of carnosine on the telomeric DNA of cultured human fetal lung fibroblast cells. Cells continuously grown in 20 mM carnosine exhibited a slower telomere shortening rate and extended lifespan in population doublings. When kept in a long-term nonproliferating state, they accumulated much less damages in the telomeric DNA when cultured in the presence of carnosine. We suggest that the reduction in telomere shortening rate and damages in telomeric DNA made an important contribution to the life-extension effect of carnosine.     

Reactive oxygen-mediated damage to a human DNA replication and repair protein

Ultraviolet A (UVA) makes up more than 90% of incident terrestrial ultraviolet radiation. Unlike shorter wavelength UVB, which damages DNA directly, UVA is absorbed poorly by DNA and is therefore considered to be less hazardous. Organ transplant patients treated with the immunosuppressant azathioprine frequently develop skin cancer. Their DNA contains 6-thioguanine-a base analogue that generates DNA-damaging singlet oxygen ((1)O(2)) when exposed to UVA. Here, we show that this (1)O(2) damages proliferating cell nuclear antigen (PCNA), the homotrimeric DNA polymerase sliding clamp. It causes covalent oxidative crosslinking between the PCNA subunits through a histidine residue in the intersubunit domain. Crosslinking also occurs after treatment with higher-although still moderate-doses of UVA alone or with chemical oxidants. Chronic accumulation of oxidized proteins is linked to neurodegenerative disorders and ageing. Our findings identify oxidative damage to an important DNA replication and repair protein as a previously unrecognized hazard of acute oxidative stress.     

DNA damage in the spleen, liver and kidneys of mice treated with ochratoxin A

Ochratoxin A a natural contaminant of feed and food has been shown to induce experimental liver and kidney tumors. Since there is a good correlation between the carcinogenic potency of chemicals and the DNA damages induced in mammalian cells treated either in vivo or in vitro by these compounds, we have measured single-strand breaks induced by ochratoxin A in DNA of liver, spleen and kidney. Our data clearly showed that ochratoxin A induced DNA damages in vitro as well as in vivo. Damages were dose-dependent, reversible and vary upon the time according to the tissue. In spite there is no report up to now on experimental leukemia induced by ochratoxin A, our results indicate that this possibility have to be considered.     

Nbs1‐mediated DNA damage repair pathway regulates haematopoietic stem cell development and embryonic haematopoiesis

ObjectivesDNA damages pose threats to haematopoietic stem cells (HSC) maintenance and haematopoietic system homeostasis. Quiescent HSCs in adult mouse bone marrow are resistant to DNA damage, while human umbilical cord blood‐derived proliferative HSCs are prone to cell death upon ionizing radiation. Murine embryonic HSCs proliferate in foetal livers and divide symmetrically to generate HSC pool. How murine embryonic HSCs respond to DNA damages is not well‐defined.Materials and methodsMice models with DNA repair molecule Nbs1 or Nbs1/p53 specifically deleted in embryonic HSCs were generated. FACS analysis, in vitro and in vivo HSC differentiation assays, qPCR, immunofluorescence and Western blotting were used to delineate roles of Nbs1‐p53 signaling in HSCs and haematopoietic progenitors.ResultsNbs1 deficiency results in persistent DNA breaks in embryonic HSCs, compromises embryonic HSC development and finally results in mouse perinatal lethality. The persistent DNA breaks in Nbs1 deficient embryonic HSCs render cell cycle arrest, while driving a higher rate of cell death in haematopoietic progenitors. Although Nbs1 deficiency promotes Atm‐Chk2‐p53 axis activation in HSCs and their progenies, ablation of p53 in Nbs1 deficient HSCs accelerates embryonic lethality.ConclusionsOur study discloses that DNA double‐strand repair molecule Nbs1 is essential in embryonic HSC development and haematopoiesis. Persistent DNA damages result in distinct cell fate in HSCs and haematopoietic progenitors. Nbs1 null HSCs tend to be maintained through cell cycle arrest, while Nbs1 null haematopoietic progenitors commit cell death. The discrepancies are mediated possibly by different magnitude of p53 signaling.