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.

[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.

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.

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.