Extreme resistance to thermally induced DNA backbone breaks in the hyperthermophilic archaeon Pyrococcus furiosus.

Pyrococcus furiosus is a hyperthermophilic archaeon that grows optimally at 100 degrees C. It is not conceivable that these organisms could survive with genomic DNA that was subject to thermal destruction, yet the mechanisms protecting the genomes of this and other hyperthermophiles against such destruction are obscure. We have determined the effect of elevated temperatures up to 110 degrees C on the molecular weight of DNA in intact P. furiosus cells, compared with the effect of elevated temperatures on DNA in the mesothermophilic bacterium Escherichia coli. At 100 degrees C, DNA in P. furiosus cells is about 20 times more resistant to thermal breakage than that in E. coli cells, and six times fewer breaks were found in P. furiosus DNA after exposure to 110 degrees C for 30 min than in E. coli DNA at 95 degrees C. Our hypothesis for this remarkable stability of DNA in a hyperthermophile is that this hyperthermophile possesses DNA-binding proteins that protect against hydrolytic damage, as well as other endogenous protective mechanisms and DNA repair enzyme systems.

Induction of slowly developing alkali-labile sites in human P3 cell DNA by UVA and blue- and green-light photons: action spectrum.

Action spectra (365-520 nm) for the formation of DNA single-strand breaks (SSB) and slowly developing alkali-labile sites (SDALS) in human teratocarcinoma P3 cells in culture were determined. Induction of SDALS results from the absorption of blue- and green-light photons. The spectrum has a broad peak that is maximal between 400 nm to 500 nm and declines sharply above and below these wavelength regions. Negligible yields of SDALS were produced by photons at wavelengths of 365 nm or shorter and at 520 nm or longer, whereas for SSB, the action ioffeases with shorter wavelength throughout the whole spectral range studied. The configuration of the SDALS action spectrum suggests that the primary chromophore, and therefore possibly the photosensitizer, is a mixture of porphyrin and flavin residues.

DNA damage produced by exposure of supercoiled plasmid DNA to high- and low-LET ionizing radiation: effects of hydroxyl radical quenchers.

Production of DNA damage by exposure to ionizing radiation was measured in two in vitro systems. A supercoiled plasmid of 7.3 kbp was isolated and exposed in an aqueous environment to 60Co gamma rays and JANUS 0.85 MeV fission-spectrum neutrons. Dose responses for the production of single-strand breaks (ssbs) and double-strand breaks (dsbs) were computed from the conversion of the supercoil to its relaxed and linear forms. The relative effectiveness (neutrons:gamma-rays) for destruction of genetic transforming activity of M13 viral DNA was 0.23, close to that for ssb production, in contrast with the situation for biological effects such as lethality, mutagenesis and cellular transformation measured in mammalian cells, where RBEs are > 1. The role of hydroxyl (OH) radicals in DNA damage induction by neutrons was investigated by exposure of plasmid in the presence of known quenchers of this species. Of four quenchers tested, all were able to reduce the yields of both ssbs and dsbs. These findings are consistent with a model for ssb and dsb induction by high linear energy transfer radiation that involves OH radical medication.

The hyperthermophilic glycolytic enzyme enolase in the archaeon, Pyrococcus furiosus: comparison with mesophilic enolases.

High enolase activity, as measured by the conversion of 2-phosphoglycerate to phosphoenolpyruvate, was found in the cytoplasm of Pyrococcus furiosus (an anaerobic, hyperthermophilic archaeon that grows optimally at 100 degrees C). In this organism, the enzyme probably functions in a sugar fermentation pathway. The enzyme was purified to homogeneity. It had a temperature optimum of > 90 degrees C and a pH optimum of 8.1. The enzyme was extremely thermostable with a time for 50% inactivation at 100 degrees C of 40 min. In contrast, an enolase from yeast was totally inactivated in 1 min at 88 degrees C. Both the P. furiosus and yeast enzymes required a metal ion for activity, but whereas the yeast enzyme has an absolute requirement for Mg2+, the P. furiosus enolase was equally active in the presence of Mn2+. Both enzymes were competitively inhibited by citrate. P. furiosus enolase, as for mesophilic enolases, probably has a homodimeric structure with subunit M(r) greater than 45,000. A highly conserved sequence of eight amino acids in the N-terminal region was found in enolases from P. furiosus and a wide range of other organisms including bacteria, yeast, birds, and mammals. Substantial differences in the thermal properties of the hyperthermophilic enzyme compared with that from less extreme thermophiles and mesophiles might be due to a substantially enhanced composition of hydrophobic amino acids.

Isolation of V79 fibroblast cell lines containing elevated metallothionein levels that have increased resistance to the cytotoxic effects of ultraviolet-A radiation.

Isolated clones of V79 Chinese hamster lung fibroblasts, selected for resistance against cadmium toxicity, were exposed to monochromatic 365 nm ultraviolet-A (UVA; 320 nm to visible light) radiation and examined for cell survival. All three of the Cd-resistant V79 clones (V79Cd) tested exhibited significant increases in survival after irradiation compared with control cultures similar to the increased survival observed in Zn acetate-induced V79 cells. Dose-modifying factors calculated for these survival experiments were all approximately 1.5. When characterized for steady-state levels of metallothionein (MT) mRNA and associated Cd-binding activity, all of the Cd-resistant V79Cd clones demonstrated elevated constitutive levels of both, implicating MT as the mechanism responsible for the observed cellular resistance to Cd and also to 365 nm UVA radiation. However, whereas levels of intracellular MT protein correlated with differences in survival against Cd, MT intracellular levels did not correlate well with protection against 365 nm UVA. Increased cell survival after exposure to 365 nm UVA radiation mediated by MT appeared to reach a threshold level and MT only provided a limited degree of protection. Since UVA radiation is known to cause cell death mediated through the intracellular generation of reactive oxygen species (ROS), these results suggest that the role of MT in ameliorating cellular photooxidative damage produced by UVA is by reducing intracellular ROS.

Dependence of the yield of strand breaks induced by gamma-rays in DNA on the physical conditions of exposure: water content and temperature.

The induction by 60Co gamma-rays of DNA breaks, revealed by relaxation (single-strand breaks, SSBs) and linearization (double-strand breaks, DSBs) of supercoiled plasmid DNA, was measured under three irradiation conditions, the DNA being in a dry, humid, or aqueous state in the absence of oxygen, at 25 or -196 degrees C (77 K). Yields of strand breaks (3.0 x 10(-10) SSB/Gy.Da and 2.6 x 10(-11) DSB/Gy.Da) in DNA exposed to a stream of humidified nitrogen were higher than those in the dry condition (5.7 x 10(-11) SSB/Gy.Da and 3.2 x 10(-12) DSB/Gy.Da), but both these yields were markedly lower than those measured for DNA in aqueous solution at a concentration of 73 micrograms/cm3 (1.14 x 10(-7) SSB/Gy.Da and 5.4 x 10(-9) DSB/Gy.Da). Over 100-fold fewer SSBs were observed in the frozen aqueous system compared with the non-frozen liquid state, whereas in the dry and humid states, freezing did not affect the yield as much. The same trend was observed for DSBs. However, the induction of SSBs was more affected than that of DSBs by freezing in the aqueous systems. An interesting reverse relationship was observed in humid systems. The observed linearity of DSB induction with radiation dose supported a single-event mechanism. A comparison of G values for humid systems revealed that the role of bound water in radiation damage becomes significant in the nonfrozen state. Based on these and other measurements of strand breaks under different conditions, the significance of bound and free water on the yields of DNA strand breaks by gamma-rays is discussed, and the relevance of these results to the in vivo situation outlined.

A 5-4 pyrimidine-pyrimidone photoproduct produced from mixtures of thymine and 4-thiouridine irradiated with 334 nm light.

The nucleoside 4-thiouridine, present in some bacterial tRNA species, is known to be a chromophore and a target for near-UV light-induced growth delay and also mediates both photoprotection and near-UV cell killing in various bacterial strains. To investigate the photoreaction of 4-thiouridine with DNA or its precursors, we irradiated aqueous mixtures of thymine and 4-thiouridine with 334 nm light and then separated photoproducts using two or more stages of reversed-phase high performance liquid chromatography. The two equally abundant major photoproducts were analyzed by UV absorbance spectrophotometry, fast-atom bombardment and electron-impact mass spectrometry, and 1H- and 13C-NMR spectroscopy, and have been identified as two diastereomers of 6-hydroxy-5-[1-(beta-D-erythro-pentofuranosyl)-4'-pyrimidin-2'- one]dihydrothymine (O6hThy[5-4]Pdo), of molecular weight = 370.32. These two diastereomers, although stable at room temperature or below, are interconvertible by heating (90 degrees C for 5 min) in aqueous solution. The possible biological significance of this photoproduct is discussed, and an application as a crosslinker for oligonucleotides to selectively block replication is suggested.

Filter elution assays for DNA damage: practical and mechanistic significance of the DNA in the filter support wash.

The alkaline and neutral (or nondenaturing) filter elution assays are popular methods for the measurement of DNA strand breakage and its repair in eukaryotic cells. In both alkaline and neutral elution, it is recommended practice to wash the filter support after removal of the filter and to analyze the DNA recovered by this procedure together with that remaining on the filter as uneluted DNA, although it is not obvious why the DNA in the filter support wash should be so interpreted. We have observed that the sum of the DNA on the filter and that recovered in the filter support wash is approximately constant when the pH of the alkaline filter elution assay for total strand breaks is increased from 12.1 to 12.6, whereas the fraction on the filter itself is markedly smaller at the higher pH. This behavior characterized DNA elution from undamaged cells, as well as from cells treated with various DNA-damaging agents. These findings are consistent with the "tug-of-war" mechanism that has been proposed for alkaline elution, but are inconsistent with the simplest mechanism of the "sieve" class. In the neutral filter elution assay for double-strand breaks, by contrast, the distribution of DNA between the filter and the filter support wash is pH-independent. This suggests that single- and double-stranded DNA segments traverse a filter by different physical mechanisms. Our observations underscore the importance of carrying out the filter support wash and the analysis of the DNA it contains as uneluted DNA in alkaline elution, while indicating that a different analysis of this DNA might be appropriate for neutral elution.

Comparison of repair of DNA double-strand breaks caused by neutron or gamma radiation in cultured human cells.

The dose-response for the induction of initial double-strand breaks (dsb) in DNA of human epithelioid cells by JANUS 0.85 MeV fission-spectrum neutrons was parabolic as assayed by a calibrated neutral filter elution technique. The relative biological effectiveness (RBE) of these neutrons relative to 60Co gamma-rays was unity. The kinetics of repair after a 60 Gy gamma-ray exposure were biphasic. About 65% of these dsb were rapidly repaired (T 1/2 of approximately 2 min), and the remainder were almost completely removed after 150 min at a slower rate (T 1/2 = 30 min). After the same dose of JANUS neutrons, the rapid repair component was markedly reduced (possibly not a significant repair component), and the bulk of the dsb were sealed more slowly (T 1/2 = 90 min). After 150 min, 25% remained unsealed. Even after a lower neutron dose (20 Gy), a proportion of the dsb were refractory to repair. Thus, unrepaired (or irreparable) dsb induced by high energy neutrons might explain the high RBE of neutrons for cell killing.

Repair of near-visible- and blue-light-induced DNA single-strand breaks by the CHO cell lines AA8 and EM9.

The induction of single-strand breaks (SSB) and the kinetics of SSB repair were measured in two Chinese hamster ovary cell lines irradiated with monochromatic photons of near-visible radiation (405 nm) and blue light (434 nm). The radiosensitive and UV-A-sensitive mutant line EM9 is known to repair SSB induced by ionizing radiation or 365-nm UV-A more slowly than the parent line AA8. At the 10% survival level, EM9 cells were 1.7- and 1.6-fold more sensitive than AA8 cells to 405 and 434 nm radiation, respectively. This sensitivity was not due to differences in induction of SSB because AA8 and EM9 cells accumulated the same number of initial breaks when irradiated at 0.5 degrees C with either 405 nm (5.9 SSB per MJ/m2) or 434 nm (5.1 SSB per MJ/m2), as measured by alkaline elution. When the cells repaired these SSB at 37 degrees C in full culture medium, biphasic repair kinetics were observed for both cell lines. In both phases of repair, EM9 cells repaired breaks induced by both wavelengths more slowly than did AA8 cells. The t1/2 values for the repair phases for 405-nm-induced SSB were 3.8 and 150 min for EM9, and 1.5 and 52 min for AA8; the corresponding values for repair of 434 nm breaks were 3.7 and 39 min for EM9, and 2.0 and 30 min for AA8. Because of this slower repair, EM9 cells left more SSB unrepaired after 90 min than did AA8 cells for both wavelengths.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA breaks caused by monochromatic 365 nm ultraviolet-A radiation or hydrogen peroxide and their repair in human epithelioid and xeroderma pigmentosum cells.

The induction and repair of DNA single-strand breaks (SSB) assayed by alkaline filter elution was compared in human epithelioid P3 and xeroderma pigmentosum (XP) cells exposed to monochromatic 365-nm UV-A radiation and H2O2. Initial yields of SSB were measured with the cells held at 0.5 degrees C during exposure. The yield from exposure to 365-nm radiation was slightly greater in XP than in P3 cells, whereas H2O2 produced more than three times as many SSB in P3 compared with XP cells. o-Phenanthroline (50 mM) markedly inhibited the yields of SSB induced in XP cells by H2O2, but had no effect on those produced by 365-nm UV-A. These results are consistent with the fact that P3 cells, unlike XP cells, have undetectable levels of catalase. The measured production of trace amounts of H2O2 by the actual 365-nm UV-A exposures was not sufficient to account for the numbers of breaks that were observed. Single-strand breaks produced by both agents were completely repaired after 50 min in P3 cells, as were H2O2-induced SSB in XP cells. However, 25% of the 365-nm UV-A-induced SSB in XP cells remained refractory to repair after 60 min. The results show that SSB produced by these two agents are different and that 365 nm radiation produces most SSB in cells by mechanisms other than by production of H2O2.

Enhanced expression of protein kinase C gene caused by solar radiation.

Natural solar radiation (5 min of midday exposure in mid July, latitude 42 degrees N) induces protein kinase C mRNA almost two-fold in human epithelioid P3 cells in culture. This response is the same as that following tumor promotion by chemicals. The result indicates a possible role of promotion by solar UV radiation.

Correlation between cell survival and DNA single-strand break repair proficiency in the Chinese hamster ovary cell lines AA8 and EM9 irradiated with 365-nm ultraviolet-A radiation.

Cell survival parameters and the induction and repair of DNA single-strand breaks were measured in two Chinese hamster ovary cell lines after irradiation with monochromatic UVA radiation of wavelength 365 nm. The radiosensitive mutant cell line EM9 is known to repair ionizing-radiation-induced single-strand breaks (SSB) more slowly than the parent line AA8. EM9 was determined to be 1.7-fold more sensitive to killing by 365-nm radiation than AA8 at the 10% survival level, and EM9 had a smaller shoulder region on the survival curve (alpha = 1.76) than AA8 (alpha = 0.62). No significant differences were found between the cell lines in the initial yields of SSB induced either by gamma-radiation (as determined by alkaline sucrose gradient sedimentation) or by 365-nm UVA (as determined by alkaline elution). For measurement of initial SSB, cells were irradiated at 0.5 degrees C to minimize DNA repair processes. Rejoining of 365-nm induced SSB was measured by irradiating cells at 0.5 degrees C, allowing them to repair at 37 degrees C in full culture medium, and then quantitating the remaining SSB by alkaline elution. The repair of these breaks followed biphasic kinetics in both cell lines. EM9 repaired the breaks more slowly (t1/2 values of 1.3 and 61.3 min) than did AA8 (t1/2 values of 0.9 and 53.3 min), and EM9 also left more breaks unrepaired 90 min after irradiation (24% vs 8% for AA8). Thus, the sensitivity of EM9 to 365-nm radiation correlated with its deficiency in repairing DNA lesions revealed as SSB in alkaline elution.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of initial yields of DNA-to-protein crosslinks and single-strand breaks induced in cultured human cells by far- and near-ultraviolet light, blue light and X-rays.

The initial yields of DNA-to-protein crosslinks (dpc) caused by ionizing and nonionizing radiations were compared, with emphasis upon values within the biological dose ranges (D0). Induction of dpc in cold (0-0.5 degrees C) human P3 teratocarcinoma cells was measured by using alkaline elution techniques after exposure to monochromatic UVC (254 nm), UVB (313 nm), UVA (365 and 405 nm), and blue light (434 nm). UVC and UVB light induced detectable numbers (about 100 dpc per cell per D0). Monochromatic UVA radiations produced yields about 8 times higher than UVC or UVB (for 365 nm, about 1500 dpc per cell per D0) Similar results at low doses were obtained for measurements of single-strand breaks induced by the different radiations. The action spectra for dpc were closely similar. The biological significance of these relatively high numbers of DNA lesions caused by environmental nonionizing radiation that readily penetrates into human skin is not understood.

Photosensitized damage to supercoiled plasmid DNA induced by 334-nm radiation in the presence of 2-thiouracil consists of alkali- and piperidine-labile sites as well as frank strand breaks.

A covalently closed, supercoiled plasmid was irradiated with 334-nm ultraviolet radiation in the presence of the naturally occurring photosensitizer 2-thiouracil (s2Ura). After irradiation, some DNA samples were treated to reveal labile sites. Agarose gel electrophoresis was then used to resolve the unrelaxed supercoils from the relaxed forms, and the DNA bands were quantitated by fluorescence scanning. Irradiation of the plasmid in the absence of s2Ura induced small numbers of frank DNA strand breaks (FSB), alkali-labile sites (ALS), and piperidine-labile sites (PLS). The induction of each of these lesions was enhanced 30 times when s2Ura was present during aerobic irradiation. Anoxia, as well as the hydroxyl radical scavengers acetate and formate, inhibited the formation of all three lesion types. The relative proportions of the three lesion types produced by several DNA damaging treatments were measured. Hydrogen peroxide, gamma-irradiation, and s2Ura photosensitization produced nearly identical damage proportions, with PLS: FSB ratios of 1.25:1, 0.78:1, and 0.84:1, respectively. Treatment with singlet oxygen [data from Blazek et al. (1989) Photochem. Photobiol. 48, 607-613] produced much different proportions, with a PLS:FSB ratio of 4.1:1. These results may indicate a role for hydroxyl radical in s2Ura-photosensitized DNA damage.

Evidence that hydrogen peroxide generated by 365-nm UVA radiation is not important in mammalian cell killing.

We compared measurements of cell survival and DNA single-strand breaks (SSBs) caused by hydrogen peroxide (H2O2) and UVA radiation (365-nm) in both a parental and a H2O2-resistant variant of the Chinese hamster ovary HA1 line derived by culturing cells in progressively higher concentrations of H2O2. Both RNA slot blot analysis and enzyme analysis confirmed that the variant possesses high levels of both catalase activity and mRNA. The variant was completely resistant to the lethal effects of H2O2 over the concentration range tested (up to 480 microM), whereas the parental strain showed less than 1% survival at this concentration. Similarly, the H2O2-resistant strain exhibited far fewer SSBs after exposure to H2O2 than the parental strain. Addition of o-phenanthroline to the parental cells during H2O2 exposure almost completely inhibited SSB induction, evidence that these SSBs are produced via the Fenton pathway of Haber-Weiss reactions. Very little difference was found between the variant and the parent after exposure to 365-nm radiation: only a minor difference in survival kinetics and no difference is SSB induction were observed between the two cell lines. These results are consistent with a hypothesis that most lethal events caused in cells by UVA occur by pathways that do not involve the H2O2 that is produced by sensitized reactions within the cells.

Ultraviolet light induces double-strand breaks in DNA of cultured human P3 cells as measured by neutral filter elution.

Neutral filter elution at pH 7.2 and 9.6 was used to measure the induction of DNA lesions in human P3 teratocarcinoma cells by monochromatic 254-, 270-, 313-, 334-, 365-, and 405-nm radiation and by 60 gamma rays. In this assay DNA double-strand breaks (dsb) increase the rate of elution of DNA from cell lysates on a filter. Yields of dsb as measured by this procedure were determined by using a calibration of the assay that correlates elution parameters with number of dsb caused by disintegration of 125I incorporated into the DNA. Analysis of fluence responses obtained by using the calibrated assay indicated that the number of dsb induced per dalton of DNA as measured by this assay is proportional to the square of the fluence at all the energies of radiation studied, implying that the induction of these lesions may be a two-hit event. Analysis of the relative efficiencies for the induction of dsb by ultraviolet radiation, corrected for quantum efficiency, revealed a spectrum that coincided closely with that for the induction of single-strand breaks (ssb) in the same cells, having a close fit with the spectrum of nucleic acid in the UVC and UVB region below 313 nm, and a shoulder in the UVA region. It was calculated, however, that there may be too few ssb for dsb to result from randomly distributed closely opposed ssb.

Induction and repair of DNA strand breaks in Bacteroides fragilis.

Alkaline sucrose gradient sedimentation was used to establish whether strand breakage and repair take place in the DNA of UV-irradiated Bacteroides fragilis during the removal of pyrimidine dimers. A B. fragilis wild-type strain and two of its repair mutants, a mitomycin C sensitive mutant (MTC25) having wild-type levels of UV survival, and a UV-sensitive, mitomycin C sensitive mutant (UVS9), were investigated. Under anaerobic conditions, far-UV irradiation induced metabolically regulated strand breakage and resynthesis in the wild-type strain, but this was markedly reduced in both the MTC25 and UVS9 mutants. Approximately half of the strand breaks generated by the various strains were rejoined during further holding in buffer. Under replicating conditions, complete repair of strand breaks in the wild type was observed. Caffeine treatment under anaerobic conditions caused direct DNA strand breakage in B. fragilis cells but did not inhibit UV-induced breakage or repair.

Hydroxyl radical quenching agents protect against DNA breakage caused by both 365-nm UVA and by gamma radiation.

The ability of hydroxyl radical (.OH) scavengers to reduce DNA breakage in isolated DNA from Bacillus subtilis by either gamma radiation or monochromatic radiation in the UVA region (365 nm) was examined by comparing dose reduction factors (the ratio of dose required to induce n DNA breaks in the absence to the presence of quencher). Previous data have demonstrated that acetate, formate, azide, and mannitol protect supercoiled DNA against gamma-radiation-induced ssb (single-strand breaks-relaxation of supercoil by first nick) in close agreement with the rate at which their solutions quench .OH. Here we show that these quenchers also protect against 365-nm-induced ssb. The ratios for protection against 365-nm induced DNA ssb in isolated B. subtilis DNA by the four quenchers are also in proportion to their ability to quench .OH. In view of the diverse chemical nature of the quenchers and the wide range of concentrations involved, these findings are evidence that both these radiations may induce ssb in DNA via a common step that might involve .OH.