Investigation of radiation damage in DNA by using atomic force microscopy.

The effect of radiations on supercoiled plasmid DNA has been investigated by using atomic force microscopy (AFM). The DNA molecules were deposited on a substrate and observed by AFM. Alternatively, DNA at different scavenger concentrations was initially exposed to different types of radiations (alpha and X rays) at various doses. After irradiation, fragments (open circular and linearised strands) were observed corresponding to single strand breaks and double strand breaks in DNA. This result indicates the capabilities of AFM for the qualitative detection of strand modifications due to irradiation. The amount of each class of topology enables a quantitative response to be determined for both types of radiation (alpha, X). A value of the radiosensitivity of DNA was obtained as a function of the scavenger concentration. Strong accordance was found between AFM results and those obtained by use of gel electrophoresis. The advantage of AFM in comparison with traditional techniques is the possibility of analysing the radiation effects on one molecule. Indeed, taking the example of alpha particles, it is shown that it is easy to measure the sizes of linear strands by AFM. Such additional or even precise results are difficult to obtain with gel electrophoresis since, in such a case, data are lost through smearing.

The complexity of radiation-induced DNA damage as revealed by exposure to cell extracts.

The rejoining of single-strand breaks (SSBs) induced in plasmid DNA in the presence of 10 mmol dm(-3) Tris scavenger by aluminum K (Al(K)) ultrasoft X rays has been compared with that for SSBs induced by gamma radiation. The Al(K) ultrasoft X rays interact to produce low-energy secondary electrons, which are thought to be the main contributors to the formation of complex damage by low-LET radiations. The rejoining of radiation-induced SSBs was investigated using human whole cell extracts. The efficiency of rejoining of SSBs induced by Al(K) ultrasoft X rays is less than that observed for gamma-ray-induced SSBs. From the similarity of the extent of rejoining of SSBs induced by gamma rays under aerobic and anaerobic conditions, the chemical nature of the stand break termini does not significantly influence SSB rejoining. A simple nick induced in plasmid DNA by gpII protein is rejoined rapidly compared with the slower rejoining processes for radiation-induced SSBs. Therefore, ligation is not rate-determining in processing radiation-induced SSBs. This study provides further evidence that nonrejoining of radiation-induced SSBs reflects the complexity of DNA damage. From comparison of the extent of rejoining of SSBs induced by different radiations, it is inferred that double-strand breaks represent only a minor component of the overall yield of complex damage.

DNA-PK activation by ionizing radiation-induced DNA single-strand breaks.

PURPOSE: To assess the ability of 60Co gamma-radiation-induced plasmid DNA single-strand breaks (gamma-ssb) to activate the DNA-dependent protein kinase (DNA-PK) in vitro. MATERIALS AND METHODS: Plasmid DNA was gamma-irradiated under aerobic conditions to yield 0-6 gamma-ssb and <0.1 double-strand breaks (dsb) per plasmid molecule. The irradiated DNA was used to stimulate DNA-PK in crude HF19 fibroblast nuclear extracts and/or purified HeLa cell DNA-PK protein, and the activation compared with that obtained with a single enzymatically generated plasmid DNA ssb (GpII endonuclease) or dsb (EcoRI endonuclease). RESULTS: Gamma-Irradiated plasmid DNA activates DNA-PK in both crude and purified preparations and the kinase activity increases linearly with dose. As significant DNA-PK activation was detectable using irradiated plasmids which contain <0.1 dsb/molecule, it was concluded that this activation is due to gamma-ssb. However, using purified DNA-PK, this activation is relatively weak as approximately 3 approximately-ssb is equivalent to one GpII-generated DNA ssb or one end of an EcoRI-generated dsb in DNA-PK assays. CONCLUSIONS: As gamma-ssb are in a approximately 20-fold excess of approximately-dsb in vivo for low LET radiation, gamma-ssb may contribute significantly to DNA-PK signalling of gamma-radiation-induced DNA damage in vivo.

Induction of DNA-protein crosslinks in Chinese hamster V79-4 cells exposed to high- and low-linear energy transfer radiation.

The induction of DNA-protein crosslinks (DPCs) in Chinese hamster V79-4 cells after irradiation under hypoxic and aerobic conditions at 277 K with 60Co gamma rays, 238Pu alpha particles and aluminum K (Al(K)) ultrasoft X rays has been determined using a nitrocellulose filter binding assay. The dose dependences for the induction of DPCs, which involves covalent linkage, are linear over the absorbed dose range used (0-400 Gy with alpha-particle and gamma radiation, 0-600 Gy with Al(K) X rays). The yield of DPCs induced under hypoxic conditions is 55, 51 and 25 DPCs per gray per cell for 60Co gamma rays, alpha particles and Al(K) X rays, respectively. The yield of DPCs is significantly reduced in the presence of oxygen by 20, 50 and 79% for 60Co gamma rays, alpha particles and Al(K) X rays, respectively. Since the mean size of the DNA attached to the protein is uniform for 60Co gamma rays and alpha particles, variations in the DNA size do not influence the yields of DPCs. Although a DPC may be considered as a complex lesion combining two macromolecules, the dependence of the yield of DPCs on LET does not reflect the ionizing density of the radiations used. Further, this dependence on LET and the effect of oxygen do not reflect the corresponding dependences determined for a variety of biological responses. From these findings and knowledge of the radiation tracks, it is proposed that DPCs induced particularly under aerobic conditions with 60Co gamma rays are formed mainly in the sparsely ionizing segments of the radiation track.

Guanine is the target for direct ionisation damage in DNA, as detected using excision enzymes.

Exposure of an aqueous, aerated solution (pH 7) of a double-stranded DNA to 193 nm light, of sufficient energy to ionise DNA, leads to selective, non-random modification at guanine in the form of frank single-strand break (ssb) and base modifications, revealed by treatment with either Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg), Escherichia coli endonuclease III (Nth) or hot piperidine treatment. There is a similar neighbouring base sequence dependence for Fpg- and Nth-sensitive damage as that previously reported for both hot alkali-labile damage and prompt ssb. Low yields of photoproducts, namely pyrimidine dimers, are also revealed using the enzyme T4 endonuclease V (T4 endo V). Although irradiation of DNA with 193 nm light causes photoionisation of all the nucleic acid bases, these results indicate that guanine is the predominant site for localisation of the oxidative damage. These findings are consistent with migration of the radical cation to 'target' damage at guanine sites.

Irradiation of DNA with 193 nm light yields formamidopyrimidine-DNA glycosylase(Fpg) protein-sensitive lesions.

Irradiation of aqueous solutions of plasmid DNA (pUC18) at pH 7.6 with 193 nm laser light results in low yields of prompt single strand breakage (air-saturated sample phi ssh = [1.5 +/- 0.1] x 10(4), argon-saturated sample phi ssh = [0.9 +/- 0.1] x 10(4). Treatment of the irradiated DNA samples with Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) protein results in an approximate 20-fold increase in the yield of single strand break-age (air-saturated sample phi fpg = [33.1 +/- 3.1] x 10(-4), argon-saturated sample phi fpg = [23.8 +/- 2.6] x 10(-4). This result indicates that 193 nm light induces other modification(s) (most likely of the purine moieties) that are 20 times more abundant than prompt strand breakage within the DNA matrix.

Assessment of the repair and damage of DNA induced by parent and reduced RSU-1069, a 2-nitroimidazole-aziridine.

The cellular repair and damage of DNA induced by parent and reduced RSU-1069, a 2-nitroimidazole-aziridine, was assessed at both the molecular and cellular level. At the molecular level, after in vitro incubation with parent or reduced RSU-1069, plasmid DNA was transfected into Escherichia coli (AB1157) with subsequent selection for gene expression. For equivalent levels of DNA strand breakage following such treatment it is evident from the relative transformation frequencies that interactions with reduced RSU-1069 lead to DNA damage consistent with bifunctional action of a metabolite(s). At the cellular level, the cytoxicity of RSU-1069 was determined for a series of repair deficient mutants of E. coli under both aerobic and hypoxic conditions. The differential aerobic:hypoxic cytotoxicity ratio is approximately 3. We conclude that the repair of cellular DNA damage induced by RSU-1069 involves activation of the gene products under the control of the recA gene and not those under the control of the ada gene. The ability of cellular systems to repair damage induced by RSU-1069 may play a significant role in determining its efficiency to act as a hypoxic cell radiosensitizer and a hypoxia selective cytotoxin.