UVA Generates Pyrimidine Dimers in DNA Directly

There is increasing evidence that UVA radiation, which makes up ∼95% of the solar UV light reaching the Earth's surface and is also commonly used for cosmetic purposes, is genotoxic. However, in contrast to UVC and UVB, the mechanisms by which UVA produces various DNA lesions are still unclear. In addition, the relative amounts of various types of UVA lesions and their mutagenic significance are also a subject of debate. Here, we exploit atomic force microscopy (AFM) imaging of individual DNA molecules, alone and in complexes with a suite of DNA repair enzymes and antibodies, to directly quantify UVA damage and reexamine its basic mechanisms at a single-molecule level. By combining the activity of endonuclease IV and T4 endonuclease V on highly purified and UVA-irradiated pUC18 plasmids, we show by direct AFM imaging that UVA produces a significant amount of abasic sites and cyclobutane pyrimidine dimers (CPDs). However, we find that only ∼60% of the T4 endonuclease V-sensitive sites, which are commonly counted as CPDs, are true CPDs; the other 40% are abasic sites. Most importantly, our results obtained by AFM imaging of highly purified native and synthetic DNA using T4 endonuclease V, photolyase, and anti-CPD antibodies strongly suggest that CPDs are produced by UVA directly. Thus, our observations contradict the predominant view that as-yet-unidentified photosensitizers are required to transfer the energy of UVA to DNA to produce CPDs. Our results may help to resolve the long-standing controversy about the origin of UVA-produced CPDs in DNA.     

Pyrimidine Dimers in the DNA of Paramecium aurelia

The production and fate of thymine-containing pyrimidine dimers in Paramecium aurelia DNA was investigated in three experimental series: production of dimers by UV irradiation, fate of dimers in the dark, and “loss of photoreactivability of dimers.” It is shown that cyclobutyl dimers are made by UV irradiation of Paramecium DNA in vivo, that because of cytoplasmic absorption the number of dimers made in DNA irradiated in vivo is much lower than in DNA irradiated in vitro, that dimers are lost from animals incubated in the dark after irradiation, and that all the dimers that remain in the animals can be destroyed by photoreactivating illumination. Since mutation induction is photoreactivable, these and previous photoreactivation data suggest that pyrimidine dimers are important in mutation induction in P. aurelia.     

DNA repair after ultraviolet irradiation of ICR 2A frog cells. Pyrimidine dimers are long acting blocks to nascent DNA synthesis.

The ability of ICR 2A frog cells to repair DNA damage induced by ultraviolet irradiation was examined. These cells are capable of photoreactivation but are nearly totally deficient in excision repair. They have the ability to convert the small molecule weight DNA made after irradiation into large molecules but do not show an enhancement in this process when the UV dose is delivered in two separate exposures separated by a 3- or 24-h incubation. Total DNA synthesis is depressed and low molecular weight DNA continues to be synthesized during pulse-labeling as long as 48 h after irradiation. The effects of pyrimidine dimer removal through exposure of UV irradiated cells to photoreactivating light indicate that dimers act as the critical lesions blocking DNA synthesis.     

DNA methylation in the fungi

A systematic study on the incidence and patterns of cytosine methylation in the fungi has been carried out by restriction and nearest-neighbor analysis of DNAs isolated from undifferentiated cells of several fungal species. With respect to DNA modification, the fungi appear to be a heterogeneous group, with a 5-methylcytosine content ranging from undetectable levels (less than or equal to 0.1% of cytosine residues methylated in 18 out of 20 species tested) to low but detectable levels (e.g. congruent to 0.2 and congruent to 0.5% of the total cytosines methylated in Sporotrichum dimorphosporum and Phycomyces blakesleeanus, respectively). In the species where it has been detected, 5-methylcytosine is located mostly at CpG doublets, and the methylated sites are clustered in long tracts (10-30 kilobase pairs) separated from essentially unmethylated regions. This methylated compartment, which comprises a small fraction (1-11%) of the total DNA, contains at least a specific set of repetitive sequences. These results contrast with the higher 5-methylcytosine content found in the fungus Physarum polycephalum and in vertebrates and higher plants.     

Repeated DNA sequences in fungi

Several fungal species, representatives of all broad groups like basidiomycetes, ascomycetes and phycomycetes, were examined for the nature of repeated DNA sequences by DNA:DNA reassociation studies using hydroxyapatite chromatography. All of the fungal species tested contained 10-20% repeated DNA sequences. There are approximately 100-110 copies of repeated DNA sequences of approximately 4 × 10⁷ daltons piece size of each. Repeated DNA sequence homoduplexes showed on average 5°C difference of T_e50 (temperature at which 50% duplexes dissociate) values from the corresponding homoduplexes of unfractionated whole DNA. It is suggested that a part of repetitive sequences in fungi constitutes mitochondrial DNA and a part of it constitutes nuclear DNA.     

Pyrimidine Oligodeoxyxylonucleotides Form Triplexes with Purine DNA in Neutral Medium

Interactions of oligonucleotides comprising (1--D-2"-deoxy-threo-pentafuranosyl)thymine and (1--D-2"-deoxy-threo-pentafuranosyl)cytosine residues (oligodeoxyxylonucleotides or OXNs) with complementary single-stranded DNA fragments were investigated. Using nondenaturing gel electrophoresis, footprinting, and melting assays, pyrimidine OXNs were shown to form triplexes with the purine DNA template, which are stable at neutral pH and comparable in heat stability with the corresponding natural polypurine–polypyrimidine DNA duplexes. In such triplexes, the N3 of cytosines in one of the OXNs are protonated. As revealed by CD spectroscopy in the 210–340 nm range, the form of the triple helix depends on the nucleotide composition and sequence of the DNA template, and is intermediate between A and B.     

Pyrimidine oligodeoxyxylonucleotides form triplexes with purine DNA in neutral media

Interactions of oligonucleotides comprising (1-beta-D-2'-deoxy-threo-pentafuranosyl)thymine and (1-beta-D-2'-deoxy-threo-pentafuranosyl)cytosine residues (oligodeoxyxylonucleotides or OXNs) with complementary single-stranded DNA fragments were investigated. Using nondenaturing gel electrophoresis, footprinting, and melting assays, pyrimidine OXNs were shown to form triplexes with the purine DNA template, which are stable at neutral pH and comparable in heat stability with the corresponding natural polypurine-polypyrimidine DNA duplexes. In such triplexes, the N3 of cytosines in one of the OXNs are protonated. As revealed by CD spectroscopy in the 210-340 nm range, the form of the triple helix depends on the nucleotide composition and sequence of the DNA template, and is intermediate between A and B.     

真菌DNA条形码技术研究进展

DNA条形码(DNA barcoding)技术作为一门新兴的物种鉴定方法以其灵敏、精确、方便和客观的优势,在动植物和微生物的分类鉴定中已经得到广泛应用.真菌鉴定中常用作标准条形码的是核核糖体DNA内转录间隔区(Internal transcribed spacer,ITS),如今也有一些新型条形码被发现和应用到实际操作中,如微条形码、ND6、EF3.本文对DNA条形码技术的产生和发展做出了总结,通过研究其在真菌中应用的实际案例分析了DNA条形码技术的优缺点及发展趋势,并指出DNA条形码技术将以全新的视角来弥补传统分类学的不足,最终实现生物自身的序列变异信息与现有形态分类学的结合.     

Unexpected conserved non-coding DNA blocks in mammals

The significance of non-coding DNA is a longstanding riddle in the study of molecular evolution. Using a comparative genomics approach, Dermitzakis and colleagues have recently shown that at least some non-coding sequence, frequently ignored as meaningless noise, might bear the signature of natural selection. If functional, it could mark a turning point in the way we think about the evolution of the genome.     

Pyrimidine 5-methyl groups influence the magnitude of DNA curvature

DNA containing short sequences of the form (dA)n.(dT)n can exhibit pronounced degrees of stable curvature of the helix axis, provided that these homooligomeric stretches are approximately in phase with the helix repeat. However, the precise origin of this effect is unknown. We have observed that pyrimidine 5-methyl groups can have a significant effect on the degree of curvature, depending on their locations within the homooligomeric sequences. Such effects are observed in both (dA)n.(dT/dU)n and (dI)n.(dC/d5meC)n sequence motifs, arguing for a general structural perturbation due to the methyl group. The current observations suggest that pyrimidine methyl groups could influence protein-DNA interactions not only through direct protein-methyl group contacts but also by methyl group induced alterations in local DNA structure.     

Osmium-Based Pyrimidine Contrast Tags for Enhanced Nanopore-Based DNA Base Discrimination

Nanopores are a promising platform in next generation DNA sequencing. In this platform, an individual DNA strand is threaded into nanopore using an electric field, and enzyme-based ratcheting is used to move the strand through the detector. During this process the residual ion current through the pore is measured, which exhibits unique levels for different base combinations inside the pore. While this approach has shown great promise, accuracy is not optimal because the four bases are chemically comparable to one another, leading to small differences in current obstruction. Nucleobase-specific chemical tagging can be a viable approach to enhancing the contrast between different bases in the sequence. Herein we show that covalent modification of one or both of the pyrimidine bases by an osmium bipyridine complex leads to measureable differences in the blockade amplitudes of DNA molecules. We qualitatively determine the degree of osmylation of a DNA strand by passing it through a solid-state nanopore, and are thus able to gauge T and C base content. In addition, we show that osmium bipyridine reacts with dsDNA, leading to substantially different current blockade levels than exhibited for bare dsDNA. This work serves as a proof of principle for nanopore sequencing and mapping via base-specific DNA osmylation.     

The DNA damage response in filamentous fungi

The mechanisms used by fungal cells to repair DNA damage have been subjects of intensive investigation for almost 50 years. As a result, the model yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae have led the way in yielding critical insights into the nature of the DNA damage response. At the same time, largely through the efforts of Etta Kafer, Hirokazu Inoue, and colleagues, a substantial collection of Aspergillus nidulans and Neurospora crassa DNA repair mutants has been identified and characterized in detail. As the analysis of these mutants continues and increasing amounts of annotated genome sequence become available, it is becoming readily apparent that the DNA damage response of filamentous fungi possesses several features that distinguish it from the model yeasts. These features are emphasized in this review, which describes the genes, regulatory networks, and processes that compose the fungal DNA damage response. Further characterization of this response will likely yield general insights that are applicable to animals and plants. Moreover, it may also become evident that the DNA damage response can be manipulated to control fungal growth.     

DNA Damage Levels Determine Cyclobutyl Pyrimidine Dimer Repair Mechanisms in Alfalfa Seedlings

Ultraviolet radiation in sunlight damages DNA in plants, but little is understood about the types, lesion capacity, and coordination of repair pathways. We challenged intact alfalfa seedlings with UV doses that induced different initial levels of cyclobutyl pyrimidine dimers and measured repair by excision and photoreactivation. By using alkaline gel electrophoresis of nonradioactive DNAs treated with a cyclobutyl pyrimidine dimer-specific UV endonuclease, we quantitated ethidium-stained DNA by electronic imaging and calculated lesion frequencies from the number average molecular lengths. At low initial dimer frequencies (less than ~30 dimers per million bases), the seedlings used only photoreactivation to repair dimers; excision repair was not significant. At higher damage levels, both excision and photorepair contributed significantly. This strategy would allow plants with low damage levels to use error-free repair requiring only an external light energy source, whereas seedlings subjected to higher damage frequencies could call on additional repair processes requiring cellular energy. Characterization of repair in plants thus requires an investigation of a range of conditions, including the level of initial damage.     

嘧啶核苷的研究进展

嘧啶核苷包括尿嘧啶核苷和胞嘧啶核苷,其在食品工业和医药行业上应用广泛。介绍了嘧啶核苷的用途、测定方法、生产方法等;根据代谢控制发酵原理,以尿苷生产菌的选育为例,详细介绍了嘧啶核苷生产菌的育种策略,并对嘧啶核苷生产菌的育种实例、育种展望进行了综述。     

Occurrence of fungi on submerged pine and teak blocks in Hong Kong coastal waters

An investigation of the lignicolous marine fungi in Hong Kong coastal waters was carried out for 18 months in order to determine successional patterns of fungal occurrence. Submerged blocks of pine (Pinus massoniana Lambert) and teak (Tectona grandis Linnaeus) were used as baits and were scraped at intervals to remove surface fouling organisms. Occurrence of fungi is described in terms of the composition of the fungal flora and the number of fungal colonies developed on the test blocks before and after incubation. The scraped teak and pine blocks supported slightly different fungal floras, and the selective effect of these substrates on fungal colonization is discussed.     

Factors Affecting the Tailing of Blunt End DNA with Fluorescent Pyrimidine dNTPs

The transferase activity of non-proofreading DNA polymerases is a well-known phenomenon that has been utilized in cloning and sequencing applications. The non-templated addition of modified nucleotides at DNA blunt ends is a potentially useful feature of DNA polymerases that can be used for selective transformation of DNA 3′ ends. In this paper, we characterized the tailing reaction at perfectly matched and mismatched duplex ends with Cy3- and Cy5-modified pyrimidine nucleotides. It was shown that the best DNA tailing substrate does not have a perfect Watson–Crick base pair at the end. Mismatched duplexes with a 3′ dC were the most efficient in the Taq DNA polymerase-catalysed tailing reaction with a Cy5-modified dUTP. We further demonstrated that the arrangement of the dye residue relative to the nucleobase notably affects the outcome of the tailing reaction. A comparative study of labelled deoxycytidine and deoxyuridine nucleotides showed higher efficiency for dUTP derivatives. The non-templated addition of modified nucleotides by Taq polymerase at a duplex blunt end was generally complicated by the pyrophosphorolysis and 5′ exonuclease activity of the enzyme.     

DNA extraction method for PCR in mycorrhizal fungi

AIMS: To develop a simple and rapid DNA extraction protocol for PCR in mycorrhizal fungi. METHODS AND RESULTS: The protocol combines the application of rapid freezing and boiling cycles and passage of the extracts through DNA purification columns. PCR amplifiable DNA was obtained from a number of endo- and ecto-mycorrhizal fungi using minute quantities of spores and mycelium, respectively. CONCLUSION: DNA extracted following the method, was used to successfully amplify regions of interest from high as well as low copy number genes. The amplicons were suitable for further downstream applications such as sequencing and PCR-RFLPs. SIGNIFICANCE AND IMPACT OF THE STUDY: The protocol described is simple, short and facilitates rapid isolation of PCR amplifiable genomic DNA from a large number of fungal isolates in a single day. The method requires only minute quantities of starting material and is suitable for mycorrhizal fungi as well as a range of other fungi.