G-quadruplex DNA structures and their relevance in radioprotection.

BACKGROUND: DNA, the genetic material of most of the organisms, is the crucial element of life. Integrity of DNA needs to be maintained for transmission of genetic material from one generation to another. All organisms are constantly challenged by the environmental conditions which can lead to the induction of DNA damage. Ionizing radiation (IR) has been known to induce DNA damage and IR sensitivity varies among different organisms. The causes for differential radiosensitivity among various organisms have not been studied in great detail. SCOPE OF REVIEW: We discuss DNA secondary structure formation, GC content of the genome, role of G-quadruplex formation, and its relationship with radiosensitivity of the genome. MAJOR CONCLUSION: In Deinococcus radiodurans, the bacterium that exhibits maximum radio resistance, multiple G-quadruplex forming motifs are reported. In human cells, G-quadruplex formation led to differential radiosensitivity. In this article, we have discussed, the role of secondary DNA structure formation like G-quadruplex in shielding the genome from radiation and its implications in understanding evolution of radio protective effect of an organism. We also discuss role of GC content and its correlation with radio resistance. GENERAL SIGNIFICANCE: This review provides an insight into the role of G-quadruplexes in providing differential radiosensitivity at different site of the genome and in different organisms. It further discusses the possibility of higher GC content contributing towards reduced radiosensitivity in different organisms, evolution of radiosensitivity, and regulation of multiple cellular processes.

The complete mitochondrial genome of Gonepteryx mahaguru (Lepidoptera: Pieridae).

The complete mitochondrial genome of Gonepteryx mahaguru (Lepidoptera: Pieridae) is 15,221 bp in length, containing 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (LrRNA and SrRNA) and 1 non-coding A + T-rich region. The nucleotide composition is significantly biased toward A + T (80.9%). All PCGs are initiated by classical ATN codon, with the exception of COI, which begins with TTA codon. Nine PCGs harbor the complete stop codon TAA, whereas COI, COII, ND4 and ND5 stop with incomplete codons, single T or TA. All tRNAs can be folded into the typical cloverleaf secondary structure, except for tRNA(Ser)(AGN). The A + T content of AT-rich region is 95.2%, same to the highest one in the known species in Pieridae.

A contact photo-cross-linking investigation of the active site of the 8-17 deoxyribozyme.

The small RNA-cleaving 8-17 deoxyribozyme (DNAzyme) has been the subject of extensive mechanistic and structural investigation, including a number of recent single-molecule studies of its global folding. Little detailed insight exists, however, into this DNAzyme's active site; for instance, the identity of specific nucleotides that are proximal to or in contact with the scissile site in the substrate. Here, we report a systematic replacement of a number of bases within the magnesium-folded DNAzyme-substrate complex with thio- and halogen-substituted base analogues, which were then photochemically activated to generate contact cross-links within the complex. Mapping of the cross-links revealed a striking pattern of DNAzyme-substrate cross-links but an absence of significant intra-DNAzyme cross-links. Notably, the two nucleotides directly flanking the scissile phosphodiester cross-linked strongly with functionally important elements within the DNAzyme, the thymine of a G.T wobble base pair, a WCGR bulge loop, and a terminal AGC loop. Mutation of the wobble base pair to a G-C pair led to a significant folding instability of the DNAzyme-substrate complex. The cross-linking patterns obtained were used to generate a model for the DNAzyme's active site that had the substrate's scissile phosphodiester sandwiched between the DNAzyme's wobble thymine and its AGC and WCGR loops.

A comparative evaluation of radiation-induced DNA damage using real-time PCR: influence of base composition.

To study the radiosensitivity of DNA segments at the open reading frame (gene) level, real-time PCR was used to analyze DNA damages induced by ionizing radiation. After irradiation (1, 3 and 5 kGy) of genomic DNA purified from Salmonella typhimurium, real-time PCR based on SYBR Green fluorescence and melting temperature was performed using various primer sets targeting the rfbJ, rfaJ, rfaB, hilD, ssrB, pipB, sopD, pduQ, eutG, oadB, ccmB and ccmA genes. The ccmA and ccmB genes, which existed as two copies on the chromosome and had a high GC content ( approximately 70%), showed much lower radiosensitivities than the other genes tested, particularly at 5 kGy; this distinctive feature was seen only when the genes were located on the chromosome, regardless of copy number. Our results reinforce the concept that gene sensitivity to ionizing radiation depends on the base composition and/or the spatial localization of the gene on the chromosome.

Effects of UV and visible radiation on DNA-final base damage.

Several mechanisms are likely to be involved in the solar radiation-mediated modifications of cellular DNA. Direct excitation of DNA bases by the UVB component (290-320 nm) of solar light gives rise, mostly through oxygen independent reactions, to the formation of dimeric pyrimidine lesions including cyclobutadipyrimidines, pyrimidine (6-4) pyrimidone photoproducts and related valence Dewar isomers. In addition, photoexcitation of cytosine and guanine may lead to the formation in relatively minor yields of 6-hydroxy-5,6-dihydrocytosine and 8-oxo-7,8-dihydroguanine, respectively. A second mechanism that requires the participation of endogenous photosensitizers together with oxygen is at the origin of most of the DNA damage generated by the UVA (320-400 nm) and visible light. Singlet oxygen, which arises from a type II mechanism, is likely to be mostly involved in the formation of 8-oxo-7,8-dihydroguanine that was observed within both isolated and cellular DNA. However, it may be expected that the latter oxidized purine lesion together with DNA strand breaks and pyrimidine base oxidation products are also generated with a lower efficiency through Fenton type reactions. A more definitive assessment of these mechanisms would require further studies aimed at the identification and quantification of the different DNA photolesions including both dimeric pyrimidine photoproducts and photooxidized lesions.

Frequent T:A-->G:C transversions in X-irradiated mouse cells.

Ionizing radiation is a known carcinogen and teratogen. However, the point mutations produced by ionizing radiation in mammalian cells have not been fully characterized. Determination of a characteristic spectrum of X-ray-induced mutations in mammalian cells could provide clues to cellular repair processes and could serve as a marker of individual exposure to radiation. Mouse fibroblasts containing in their genome multiple copies of a recoverable lambda phage shuttle vector were used to detect and analyze radiation-induced point mutations in the supF mutation reporter gene. Following fractionated doses of ionizing radiation, a unique mutational spectrum notable for a high proportion of T:A-->G:C transversions (57%) was found. This pattern was distinct from the spectra of UV-induced and spontaneous mutations detected in the same mouse cell assay system (mainly C:G-->T:A transitions). The predominance of T:A-->G:C transversions and the pattern of mutation hot-spots are consistent with a possible role for polymerase beta in the repair of X-ray-damaged DNA. These results may also help to define a distinctive mutational signature of X-ray exposure in mammalian cells.

[Defects in the secondary structure of DNA recognizable by nuclease S1 and their possible role in the aging of mammalian somatic cells]. / Defekty vtorichnoi struktury DNK, opoznavaemye nukleazoi S1, i ikh vozmozhnanaia rol' v starenii somaticheskikh kletok mlekopitaiushchikh.

The amount of defects in DNA secondary structure recognized by nuclease S1 and their localization by sequences of various nucleotide composition were studied in relation to the age factor. It was established that a significant twofold increase in defects of DNA secondary structure was observed in hepatic cells of very old (30 months and older) intact mice only, as well as in radiation-induced acceleration of aging at the age 19 months. The prevailing localization of the defects of DNA secondary structure has been demonstrated in those sequences which were enriched with AT pairs by 3% (as compared with the average level). It was concluded that the mentioned defects of DNA secondary structure are not the cause of aging. Nevertheless they can play an essential role in the process of irreversible destruction of genome in the terminal phase of aging.

[The radiation modification of the sugar fragment in DNA: the formation of breaks, a change in polymer conformation and the transfer of the damage to the base]. / Radiatsionnaia modifikatsiia sakharnogo fragmenta v DNK: obrazovanie razryvov, izmenenie konformatsii polimera, peredacha povrezhdeniia na osnovanie.

Comparative analysis of the author's own data and data reported in the literature on the nature of the intermediate and end molecular products of radiolysis of DNA, its precursors and substances stimulating certain DNA fragments, allows to attribute the formation of breaks and alkaline-labile sites in DNA strands and changes in the polymer conformation, to the formation and transformations of some types of primary radicals of the sugar fragment. In order to explain certain effects induced by irradiation of DNA and its precursors (a balance of basic products of radiation destruction of DNA and ESR data concerning low temperature radiolysis of bases, nucleotides and nucleosides) the author proposes a model of the transfer of a damage (free valence) from 2-deoxyribosyl to a base within one nucleotide.

Suppression of cyclobutane and mean value of 6-4 dipyrimidines formation in triple-stranded H-DNA.

We have determined the effect of H-DNA formation on the distributions of two ultraviolet (UV) light induced photoproducts--cyclobutane dipyrimidines and mean value of 6-4 dipyrimidines. A region of DNA containing the sequence (dT-dC)18.(dA-dG)18 was treated under conditions that specifically yield the triple-stranded H-y3 or H-y5 DNA structure and then irradiated with UV. The positions of cyclobutane dipyrimidines and mean value of 6-4 dipyrimidines were determined by T4 endonuclease V cleavage and by hot piperidine cleavage, respectively. Formation of H-DNA structures greatly decreased the photoproduct yield in the (dT-dC)18.(dA-dG)18 region but not elsewhere in the DNA. Suppression of photoproduct formation is greater in half of the repeat, reflecting whether the DNA is in the H-y3 or H-y5 conformation. Within the repeat, the suppression was less in the middle and toward the ends. Models for the suppression of photoproduct formation in H-DNA and the possible utility of our findings are discussed.

The degree of ultraviolet light damage to DNA containing iododeoxyuridine or bromodeoxyuridine is dependent on the DNA sequence.

The sequence selectivity of 300 nm ultraviolet light damage to DNA containing bromodeoxyuridine or iododeoxyuridine was examined on DNA sequencing gels. This was accomplished using a system where an M13 template was employed to direct synthesis of DNA in which thymidine was fully substituted with bromodeoxyuridine or iododeoxyuridine. The sites of damage corresponded to the positions of analogue incorporation. The extent of damage varied considerably at different sites of cleavage and ranged from the undetectable to over fifteen times the limit of detection (as assessed by laser densitometer scans). Strong damage sites had the "consensus" sequence CTT while sites of no detectable damage had the "consensus" sequence GTR. Bromodeoxyuridine and iododeoxyuridine had the same sites of damage although the extent of damage varied at different sites and bromodeoxyuridine damage was slightly greater than iododeoxyuridine. DNA containing thymidine was not damaged to any detectable level in this system with 300 nm ultraviolet light. The use of three closely related DNA sequences as targets for damage confirmed that (1) the sites of analogue incorporation are the cause of ultraviolet damage; and (2) that the neighbouring DNA sequence is an important parameter in determining the extent of damage. It is proposed that the microstructure of DNA--in particular the distance between the 5-carbon of the pyrimidine base (which is attached to the halogen) and hydrogen on the 2' carbon of the 5'-deoxyribose--ultimately determines the degree of cleavage with large distances giving a small degree of damage and smaller distances a large degree of damage.

Detection of specific DNA sequences using antibodies recognizing UV-labelled DNA.

This non-isotopic method for detection of nucleic acids is based on the in situ labelling of the nucleic acid by exposure to UV-irradiation. The different UV-induced photoproducts, mainly of the thymidine dimer type, are recognized by purified rabbit antibodies specific to the lesions introduced. The UV-labelled nucleic acids can then be visualized by conventional immunostaining procedures. A major advantage of the technique is the low cost and the ease by which the DNA is specifically labelled. The purified rabbit antibodies were shown to be specific for UV-irradiated DNA, and the method was applied for detection of specific DNA sequences hybridized to homologous target DNA on membrane support. We believe that the sensitivity of the method can be improved, and the significance of using different UV-doses, immunostaining methods and membrane types is discussed.

[Fluorescence characteristics of dynamic changes in the DNA structure of peripheral blood cells in irradiated rats]. / Fluorestsentnaia kharakteristika dinamiki izmeneniia struktury DNK kletok perifericheskoi krovi obluchennykh krys.

A simple approach is proposed to determine locally denatured sites and stability characteristics of secondary DNA structure. The method is based on the analysis of the initial part of melting curve and the determination of changes in the optical density of DNA after heating up to a fixed temperature. The potentiality of the approach is illustrated by the experiments with DNA containing defects in the secondary structure caused by gamma-irradiation in vitro. The sensitivity of the method is less than 0.2 Gy.

[Age-related structural and functional changes in the DNA of human lymphocytes]. / Altersabhängige strukturelle und funktionelle Veränderungen an der DNA menschlicher Lymphozyten.

Characteristic changes of DNA winding and repair are detected in human peripheral lymphocytes with advancing donor age between 20 and 90 years, suggesting a progressively undifferentiated cell state. From a model approach it appears likely that the existence of immature lymphocytes favors aging as a general dysdifferentiation of somatic cells.

[Nucleotide composition and the level of DNA homology in a number of Bacillus diasticus strains]. / Nukleotidnyi sostav i uroven' gomologii DNK riada shtammov Bacillus diastcus.

The base composition of DNA and DNA homology of a number of strains Bacillus diastaticus differing in the intensity of the amilase synthesis and some phenotipic properties have been studied. The differences in the base composition of DNA have not been found. All stains studied are characterized by the high rate of DNA homology. B. diastaticus and B. stearothermophilus have been established to be genetically similar.

The use of capillary gas chromatography-mass spectrometry for identification of radiation-induced DNA base damage and DNA base-amino acid cross-links.

Application of capillary gas chromatography-mass spectrometry (GC-MS) to isolation and identification of radiation-induced DNA base damage including DNA base-amino acid crosslinks is reported. gamma-Irradiated samples of thymine (thy), thymidine (dT), thymidine-5'-monophosphate (pdT), cytosine(cyt),2'-deoxycytidine (dC), 2'-deoxycytidine-5'-monophosphate (pdC), and mixtures of thy, dT and pdT with tyrosine were used as model systems. Trimethylsilylation was used as the derivatization method. Samples containing nucleosides and nucleotides were first subjected to hydrolysis with formic acid or hydrochloric acid to remove sugar or sugar-phosphate moieties, then trimethylsilylated and analyzed by GC-MS. Trimethylsilyl derivatives of radiation-induced monomeric and dimeric products of the model systems mentioned above were shown to have excellent GC properties and easily interpretable mass spectra. The presence of the molecular ion (M+.) and a characteristic (M-CH3)+ ion in the mass spectra facilitated structural elucidation. The complementary use of tert.-butyldimethylsilyl derivatives was also demonstrated. These derivatives provided an intense characteristic (M-57)+ ion in their mass spectra, which may be used to corroborate the molecular weight and to monitor the corresponding compounds in a complex mixture by means of selected-ion monitoring. All gas chromatograms and mass spectra obtained are discussed in detail.

[Analysis of DNA degradation in the thymocytes of irradiated rats by flow cytometry]. / Analiz degradatsii DNK v timotsitakh obluchennykh krys metodom protochnoi tsitometrii.

The method of flow cytofluorometry of cells treated with probes specifically bound to AT- or GC-pairs of DNA was used to study DNA degradation in thymocytes of irradiated and hydrocortisone-treated rats. Death of thymocytes was shown to be accompanied by the decrease in the DNA content. The main regularities in the formation and accumulation of cells, the DNA content of which being lower than that of diploid cells, were the same as those of the internucleosome DNA fragmentation.

Further evidence for the DNA base specificity of light-induced banding.

Experiments have been carried out to investigate the DNA base specificity of light-induced banding (LIB) produced by photo-oxidation of chromosomes followed by Acridine Orange staining to detect denatured DNA. Nuclei of different base composition, human and onion, and fluorochromes of different base specificities and modes of binding to DNA were used. Our results indicate that specific destruction of guanine residues is the main effect of photo-oxidation under the conditions used, and that LIB is a base-specific phenomenon. In addition, photo-oxidation may also cause DNA-protein cross-linking which affects the binding of some dyes, while prolonged photo-oxidation appears to cause more general damage to DNA.

Inhibition of DNA-ethidium bromide intercalation due to free radical attack upon DNA. II. Copper(II)-catalysed DNA damage by O.2-.

The fluorescent intercalation complex of ethidium bromide with DNA was used as a probe to demonstrate damage in the base-pair region of DNA, due to the action of superoxide radicals. The O.2- radical itself, generated by gamma-radiolysis of oxygenated aqueous Na-formate solutions, is rather ineffective with respect to impairment of DNA. Copper(II) ions, known to interact with DNA by coordinate binding at purines, enhance the damaging effect of O.2-. Addition of H2O2 to the DNA/Cu(II) system gives rise to further enhancement, so that DNA impairment by O.2- becomes comparable to that initiated by .OH radicals. These results suggest that the modified, Cu(II)-catalysed, Haber-Weiss process transforms O.2- into .OH radicals directly at the target molecule, DNA-Cu2+ + O.2-----DNA-Cu+ + O2 DNA-Cu+ + H2O2----DNA...OH + Cu2+ + OH- in a "site-specific" mechanism as proposed for other systems (Samuni et al. 1981; Aronovitch et al. 1984). Slow DNA decomposition also occurs without gamma-irradiation by autocatalysis of DNA/Cu(II)/H2O2 systems. In this context we observed that Cu(II) in the DNA-Cu2+ complex (unlike free Cu2+) is capable of oxidizing Fe(II) to Fe(III), thus the redox potential of the Cu2+/Cu+ couple appears to be higher than that of the Fe3+/Fe2+ couple when the ions are complexed with DNA. Metal-catalysed DNA damage by O.2- also occurs with Fe(III), but not with Ag(I) or Cd(II) ions. It was also observed that Cu(II) ions (but neither Ag(I) nor Cd(II] efficiently quench the fluorescence of the intercalation complex of ethidium bromide with DNA.

Inhibition of DNA-ethidium bromide intercalation due to free radical attack upon DNA. I. Comparison of the effects of various radicals.

The fluorescent intercalation complex of ethidium bromide (ETB) with DNA was used as a probe to compare the effects of various radicals with respect to impairment of the DNA base-pair region. .OH radicals inhibit up to 0.7 dye intercalations per .OH at low salt concentration, and for various oxidizing species the effect decreases in the order .OH greater than Br.2- greater than N.3 much greater than (SCN).2-. DNA impairment by the .OH product of Met-Gly is comparable to that of N.3, but no effect was found due to the interaction between DNA and Lys-Tyr-Lys phenoxyl radicals. The reducing species e-aq, H., O.2-, and CO.2- hardly affect the DNA-ETB intercalation.

UV-induced reversion of his4 frameshift mutations in rad6, rev1, and rev3 mutants of yeast.

The UV-induced reversion of two his4 frameshift alleles was much reduced in rad6 mutants of Saccharomyces cerevisiae, an observation that is consistent with the hypothesis that RAD6 function is required for the induction of all types of genetic alteration in misrepair mutagenesis. The reversion of these his4 alleles, together with two others of the same type, was also reduced in rev1 and rev3 mutant strains; in these, however, the extent of the reduction varied considerably with test allele used, in a manner analogous to the results in these strains for base repair substitution test alleles. The general features of UV-induced frameshift and substitution mutagenesis therefore appear quite similar, indicating that they may depend on related processes. If this conclusion is correct, greater attention must be given to integrating models which account for the production of nucleotide additions and deletions into those concerning misrepair mutagenesis.