Comet-FISH for the evaluation of plant DNA damage after mutagenic treatments

The aim of this study was to perform a comparative investigation of the actions of three mutagens that are widely used in plant mutagenesis using the comet-FISH technique. The comet-FISH technique was used for the analysis of DNA damage and the kinetics of repair within specific DNA sequences. FISH with rDNA and telomeric/centromeric DNA probes was applied to comets that were obtained from an alkaline/neutral comet assay. Migration within specific DNA sequences was analysed after treatment with two chemical mutagens-maleic hydrazide (MH) and N-nitroso-N-methylurea (MNU), and γ-rays. Barley was used as a model plant in this study. The possible utility of specific DNA sequences in a comparative assessment of the distribution of DNA damage within a plant genome was evaluated. This study proved that the comet-FISH technique is suitable for a detailed quantification of DNA damage and repair within specific DNA sequences in plant mutagenesis. The analysis of FISH signals demonstrated that the involvement of specific DNA sequences in DNA damage was different and was dependent on the mutagen used. We showed that 5S rDNA and telomeric DNA sequences are more sensitive to mutagenic treatment, which was expressed by a stronger fragmentation and migration in comparison to the other probes used in the study. We found that 5S rDNA and telomeric DNA probes are more suitable for testing the genotoxicity of environmental factors. A comparison of the involvement of specific chromosome domains in direct DNA breakage/repair and in chromosome aberration formation after mutagen treatment indicates the compatibility of the results.     

Cohesin and DNA damage repair

Replicated DNA molecules are physically connected by cohesin complexes from the time of their synthesis in S-phase until they are segregated during anaphase of the subsequent mitosis or meiosis. This sister chromatid cohesion is essential for the biorientation of chromosomes on the mitotic or meiotic spindle. In addition, cohesion is also essential during G2-phase of the cell cycle to allow repair of DNA double-strand breaks by homologous recombination. Although cohesion can normally only be established during S-phase, recent work in yeast has shown that DNA double-strand breaks induce the recruitment of cohesin to the damage site and lead to the de novo formation of cohesion at this site. It is unknown if similar mechanisms operate in higher eukaryotes, but in mammalian cells phosphorylation of the cohesin subunit Smc1 by the protein kinase Atm has been shown to be important for DNA repair. We discuss how cohesin and sister chromatid cohesion might facilitate the repair of damaged DNA.     

SUMO化修饰与DNA损伤修复

蛋白质的翻译后修饰在很大程度上决定了蛋白质的活性、细胞定位、稳定性及蛋白质之间的相互作用.而在DNA损伤修复过程中,通过调控不同修复蛋白的翻译后修饰来影响他们的活性及细胞定位,进而导致DNA损伤修复途径的不同和修复结果的差异.新近研究表明,蛋白质的SUMO化修饰在DNA损伤修复和基因组稳定性的维护方面发挥重要作用.本文将对SUMO化修饰对DNA损伤修复的调控的最新研究进展做一综述.     

DNA damage and repair: consequences on dose-responses

Damage to DNA is considered to be the main initiating event by which genotoxins cause hereditary effects and cancer. Single or double strand breaks, bases modifications or deletions, intra- or interstrand DNA-DNA or DNA-protein cross-links constitute the major lesions formed in different proportions according to agents and to DNA sequence context. They can result in cell death or in mutational events which in turn may initiate malignant transformation. Normal cells are able to repair these lesions with fidelity or by introducing errors. Base excision (BER) and nucleotide excision (NER) repair are error-free processes acting on the simpler forms of DNA damage. A specialized form of BER involves the removal of mismatched DNA bases occurring as errors of DNA replication or from miscoding properties of damaged bases. Severe damage will be repaired according to several types of recombinational processes: homologous, illegitimate and site-specific recombination pathways. The loss of repair capacity as seen in a number of human genetic diseases and mutant cell lines leads to hypersensitivity to environmental agents. Repair-defective cells show qualitative (mutation spectrum) and quantitative alterations in dose-effect relationships. For such repair-deficient systems, direct measurements at low doses are possible and the extrapolation from large to low doses fits well with the linear or the linear-quadratic no-threshold models. Extensive debate still takes place as to the shape of the dose-response relationships in the region at which genetic effects are not directly detectable in repair-proficient normal cells. Comparison of repair mutants and wild-type organisms pragmatically suggests that, for many genotoxins and tissues, very low doses may have no effect at all in normal cells.     

Homologous recombination in DNA repair and DNA damage tolerance

Homologous recombination （HR） comprises a series of interrelated pathways that function in the repair of DNA double-stranded breaks （DSBs） and interstrand crosslinks （ICLs）. In addition, recombination provides critical support for DNA replication in the recovery of stalled or broken replication forks, contributing to tolerance of DNA damage. A central core of proteins, most critically the RecA homolog Rad51, catalyzes the key reactions that typify HR： homology search and DNA strand invasion. The diverse functions of recombination are reflected in the need for context-specific factors that perform supplemental functions in conjunction with the core proteins. The inability to properly repair complex DNA damage and resolve DNA replication stress leads to genomic instability and contributes to cancer etiology. Mutations in the BRCA2 recombination gene cause predisposition to breast and ovarian cancer as well as Fanconi anemia, a cancer predisposition syndrome characterized by a defect in the repair of DNA interstrand crosslinks. The cellular functions of recombination are also germane to DNA-based treatment modalities of cancer, which target replicating cells by the direct or indirect induction of DNA lesions that are substrates for recombination pathways. This review focuses on mechanistic aspects of HR relating to DSB and ICL repair as well as replication fork support.     

Genotoxic stress in plants: shedding light on DNA damage, repair and DNA repair helicases

Plant cells are constantly exposed to environmental agents and endogenous processes that inflict damage to DNA and cause genotoxic stress, which can reduce plant genome stability, growth and productivity. Plants are most affected by solar UV-B radiation, which damage the DNA by inducing the formation of two main UV photoproducts such as cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). Reactive oxygen species (ROS) are also generated extra- or intra-cellularly, which constitute yet another source of genotoxic stress. As a result of this stress, the cellular DNA-damage responses (DDR) are activated, which transiently arrest the cell cycle and allow cells to repair DNA before proceeding into mitosis. DDR requires the activation of Ataxia telangiectasia-mutated (ATM) and Rad3-related (ATR) genes, which regulate the cell cycle and transmit the damage signals to downstream effectors of cell-cycle progression. Since genomic protection and stability are fundamental to ensure and sustain plant diversity and productivity, therefore, repair of DNA damages is essential. In plants the bulky DNA lesions, CPDs and 6-4PPs, are repaired by a simple and error-free mechanism: photoreactivation, which is a light-dependent mechanism and requires CPD or 6-4PP specific photolyases. In addition to this direct repair process, the plants also have sophisticated light-independent general repair mechanisms, such as the nucleotide excision repair (NER) and base excision repair (BER). The completed plant genome sequences reveal that most of the genes involved in NER and BER are present in higher plants, which suggests that the network of in-built DNA-damage repair mechanisms is conserved. This article describes the insight underlying the DNA damage and repair pathways in plants. The comet assay to measure the DNA damage and the role of DNA repair helicases such as XPD and XPB are also covered.     

Radiation-induced DNA damage and its repair

Application of modern methods of organic chemistry and recombinant DNA technologies has provided new insights in the field of DNA radiation damage and its repair. An overview of the chemical nature of the lesions inflicted on DNA by ionizing radiation is presented. The structures of 29 different DNA modified base or sugar residues are shown in comprehensive formation schemes. A fraction of radiation-induced modified bases is spontaneously released from the DNA chain during irradiation. Another part remains attached to the DNA chain backbone and for its characterization mild formic acid or enzymatic hydrolysis have been used. Starting from the chemical formulae of the altered base residues, the specific repair enzymes and their modes of action are discussed. Various glycosylases and endonucleases have been purified to homogeneity, and in some cases the gene which encodes the protein cloned. Using methods derived from Maxam and Gilbert sequencing procedures and DNA fragment 32P-labelled at one end, it has been shown that the alkali-labile sites in DNA induced by radiation are strongly dependent on the DNA base sequence. Enzymatic methods have been used to analyse the DNA base defects produced by gamma-irradiation of cells under in vivo conditions. Structures of modified bases were the same as those observed when DNA was irradiated in aqueous solution.     

DNA damage checkpoint and repair centers

In eukaryotes, recombinational repair is choreographed by multiprotein complexes that are organized into focal assemblies. These foci are highly dynamic giga-dalton structures capable of simultaneously repairing multiple DNA lesions. Moreover, the composition of these repair centers depends on the nature of the DNA lesion and is tightly coordinated with progression of the cell cycle. Components of DNA repair centers are regulated by post-translational modifications such as phosphorylation, ubiquitination and sumoylation. Repair foci progress through four distinct stages: first, DNA damage recognition and binding of DNA ends by the Mre11 complex and Ku70/80; second, end-processing and binding of single-stranded DNA by replication protein A, which recruits checkpoint proteins; third, recombinational repair during S and G(2) phase; and fourth, disassembly of foci and resumption of the cell cycle.     

Mismatch repair and DNA damage signalling

Postreplicative mismatch repair (MMR) increases the fidelity of DNA replication by up to three orders of magnitude, through correcting DNA polymerase errors that escaped proofreading. MMR also controls homologous recombination (HR) by aborting strand exchange between divergent DNA sequences. In recent years, MMR has also been implicated in the response of mammalian cells to DNA damaging agents. Thus, MMR-deficient cells were shown to be around 100-fold more resistant to killing by methylating agents of the S(N)1type than cells with functional MMR. In the case of cisplatin, the sensitivity difference was lower, typically two- to three-fold, but was observed in all matched MMR-proficient and -deficient cell pairs. More controversial is the role of MMR in cellular response to other DNA damaging agents, such as ionizing radiation (IR), topoisomerase poisons, antimetabolites, UV radiation and DNA intercalators. The MMR-dependent DNA damage signalling pathways activated by the above agents are also ill-defined. To date, signalling cascades involving the Ataxia telangiectasia mutated (ATM), ATM- and Rad3-related (ATR), as well as the stress-activated kinases JNK/SAPK and p38alpha have been linked with methylating agent and 6-thioguanine (TG) treatments, while cisplatin damage was reported to activate the c-Abl and JNK/SAPK kinases in MMR-dependent manner. MMR defects are found in several different cancer types, both familiar and sporadic, and it is possible that the involvement of the MMR system in DNA damage signalling play an important role in transformation. The scope of this article is to provide a brief overview of the recent literature on this subject and to raise questions that could be addressed in future studies.     

DNA repair: models for damage and mismatch recognition

Maintaining the integrity of the genome is critical for the survival of any organism. To achieve this, many families of enzymatic repair systems which recognize and repair DNA damage have evolved. Perhaps most intriguing about the workings of these repair systems is the actual damage recognition process. What are the chemical characteristics which are common to sites of nucleic acid damage that DNA repair proteins may exploit in targeting sites? Importantly, thermodynamic and kinetic principles, as much as structural factors, make damage sites distinct from the native DNA bases, and indeed, in many cases, these are the features which are believed to be exploited by repair enzymes. Current proposals for damage recognition may not fulfill all of the demands required of enzymatic repair systems given the sheer size of many genomes, and the efficiency with which the genome is screened for damage. Here we discuss current models for how DNA damage recognition may occur and the chemical characteristics, shared by damaged DNA sites, of which repair proteins may take advantage. These include recognition based upon the thermodynamic and kinetic instabilities associated with aberrant sites. Additionally, we describe how small changes in base pair structure can alter also the unique electronic properties of the DNA base pair pi-stack. Further, we describe photophysical, electrochemical, and biochemical experiments in which mismatches and other local perturbations in structure are detected using DNA-mediated charge transport. Finally, we speculate as to how this DNA electron transfer chemistry might be exploited by repair enzymes in order to scan the genome for sites of damage.     

Molecular mechanisms of DNA damage and repair: progress in plants

Despite stable genomes of all living organisms, they are subject to damage by chemical and physical agents in the environment (e.g., UV and ionizing. radiations, chemical mutagens, fungal and bacterial toxins, etc.) and by free radicals or alkylating agents endogenously generated in metabolism. DNA is also damaged because of errors during its replication. The DNA lesions produced by these damaging agents could be altered base, missing base, mismatch base, deletion or insertion, linked pyrimidines, strand breaks, intra- and inter-strand cross-links. These DNA lesions could be genotoxic or cytotoxic to the cell. Plants are most affected by the UV-B radiation of sunlight, which penetrates and damages their genome by inducing oxidative damage (pyrimidine hydrates) and cross-links (both DNA protein and DNA-DNA) that are responsible for retarding the growth and development. The DNA lesions can be removed by repair, replaced by recombination, or retained, leading to genome instability or mutations or carcinogenesis or cell death. Mostly organisms respond to genome damage by activating a DNA damage response pathway that regulates cell-cycle arrest, apoptosis, and DNA repair pathways. To prevent the harmful effect of DNA damage and maintain the genome integrity, all organisms have developed various strategies to either reverse, excise, or tolerate the persistence of DNA damage products by generating a network of DNA repair mechanisms. A variety of different DNA repair pathways have been reported that include direct reversal, base excision repair, nucleotide excision repair, photoreactivation, bypass, double-strand break repair pathway, and mismatch repair pathway. The direct reversal and photoreactivation require single protein, all the rest of the repair mechanisms utilize multiple proteins to remove or repair the lesions. The base excision repair pathway eliminates single damaged base, while nucleotide excision repair excises a patch of 25- to 32-nucleotide-long oligomer, including the damage. The double-strand break repair utilizes either homologous recombination or nonhomologous endjoining. In plant the latter pathway is more error prone than in other eukaryotes, which could be an important driving force in plant genome evolution. The Arabidopsis genome data indicated that the DNA repair is highly conserved between plants and mammals than within the animal kingdom, perhaps reflecting common factors such as DNA methylation. This review describes all the possible mechanisms of DNA damage and repair in general and an up to date progress in plants. In addition, various types of DNA damage products, free radical production, lipid peroxidation, role of ozone, dessication damage of plant seed, DNA integrity in pollen, and the role of DNA helicases in damage and repair and the repair genes in Arabidopsis genome are also covered in this review.     

DNA repair: bioinformatics helps reverse methylation damage

Recent work has uncovered a novel DNA repair enzyme: the AlkB protein of Escherichia coli, which oxidises the methyl groups of 1-methyladenine and 3-methylcytosine to hydroxymethyl moieties; the oxidised groups are subsequently released as formaldehyde, regenerating the unmodified bases.     

Peptide repair of oxidative DNA damage

Guanyl radical species are produced in DNA by electron removal caused by ionizing radiation, photoionization, oxidation, or photosensitization. DNA guanyl radicals can be reduced by electron donation from mild reducing agents. Important biologically relevant examples are the redox active amino acids cysteine, cystine, methionine, tryptophan, and tyrosine. We have quantified the reactivity of derivatives of these amino acids with guanyl radicals located in plasmid DNA. The radicals were produced by electron removal using the single electron oxidizing agent (SCN)(2)(*)(-). Disulfides (cystine) are unreactive. Thioethers (methionine), thiols (cysteine), and phenols (tyrosine) react with rate constants in the range 10(4)-10(6), 10(5)-10(6), and 10(5)-10(6) dm(3) mol(-1) s(-1), respectively. Indoles (tryptophan) are the most reactive with rate constants of 10(7)-10(8) dm(3) mol(-1) s(-1). Selenium analogues of amino acids are over an order of magnitude more reactive than their sulfur equivalents. Increasing positive charge is associated with a ca. 10-fold increase in reactivity. The results suggest that amino acid residues located close to DNA (for example, in DNA binding proteins such as histones) might participate in the repair of oxidative DNA damage.     

The repair of G-quadruplex-induced DNA damage

G4 DNA motifs, which can form stable secondary structures called G-quadruplexes, are ubiquitous in eukaryotic genomes, and have been shown to cause genomic instability. Specialized helicases that unwind G-quadruplexes in vitro have been identified, and they have been shown to prevent genetic instability in vivo. In the absence of these helicases, G-quadruplexes can persist and cause replication fork stalling and collapse. Translesion synthesis (TLS) and homologous recombination (HR) have been proposed to play a role in the repair of this damage, but recently it was found in the nematode Caenorhabditis elegans that G4-induced genome alterations are generated by an error-prone repair mechanism that is dependent on the A-family polymerase Theta (Pol θ). Current data point towards a scenario where DNA replication blocked at G-quadruplexes causes DNA double strand breaks (DSBs), and where the choice of repair pathway that can act on these breaks dictates the nature of genomic alterations that are observed in various organisms.     

Mitochondrial DNA damage and repair in neurodegenerative disorders

By producing ATP and regulating intracellular calcium levels, mitochondria are vital for the function and survival of neurons. Oxidative stress and damage to mitochondrial DNA during the aging process can impair mitochondrial energy metabolism and ion homeostasis in neurons, thereby rendering them vulnerable to degeneration. Mitochondrial abnormalities have been documented in all of the major neurodegenerative disorders-Alzheimer's, Parkinson's and Huntington's diseases, and amyotrophic lateral sclerosis. Mitochondrial DNA damage and dysfunction may be downstream of primary disease processes such as accumulation of pathogenic proteins. However, recent experimental evidence demonstrates that mitochondrial DNA damage responses play important roles in aging and in the pathogenesis of neurodegenerative diseases. Therapeutic interventions that target mitochondrial regulatory systems have been shown effective in cell culture and animal models, but their efficacy in humans remains to be established.